Publications

The blood enzyme glutamate-oxaloacetate transaminase (GOT) has been postulated as an effective therapeutic to protect the brain during stroke. To demonstrate its potential clinical utility, a new human recombinant form of GOT (rGOT) was produced for medical use. We tested the pharmacokinetics and evaluated the protective efficacy of rGOT in rodent and non-human primate models that reflected clinical stroke conditions. We found that continuous intravenous administration of rGOT within the first 8 h after ischemic onset significantly reduced the infarct size in both severe (30%) and mild lesions (48%). Cerebrospinal fluid and proteomics analysis, in combination with positron emission tomography imaging, indicated that rGOT can reach the brain and induce cytoprotective autophagy and induce local protection by alleviating neuronal apoptosis. Our results suggest that rGOT can be safely used immediately in patients suspected of having a stroke. This study requires further validation in clinical stroke populations.

Glutamate oxaloacetate transaminase 1 (GOT1) enzyme plays a critical role in the cell metabolism by participating in the carbohydrate and amino acid metabolism. In ischemic stroke, we have demonstrated that recombinant GOT1 acts as a novel neuroprotective treatment against the excess of extracellular glutamate that accumulates in the brain following ischemic stroke. In this study, we investigated the inhibitory effect of GOT1 on brain metabolism and on the ischemic damage in a rat model of ischemic stroke by means of a specific antibody developed against this enzyme. Inhibition of GOT1 caused higher brain glutamate and lactate levels and this response was associated with larger ischemic lesion. This study represents the first demonstration that the inhibition of the blood GOT1 activity leads to more severe ischemic damage and poorer outcome and supports the protective role of GOT1 against ischemic insults.

Organophosphate (OP) based pesticides are highly toxic compounds that are still widely used in agriculture around the world. According to World Health Organization (WHO) data, it is estimated that between 250,000 and 370,000 deaths occur yearly around the globe as a result of acute intoxications by pesticides. Currently available antidotal drug treatments of severe OP intoxications are symptomatic, do not reduce the level of intoxicating OP in the body and have limited ability to prevent long-term brain damage. Pesticide poisonings present a special therapeutic challenge since in many cases, such as with parathion, their toxicity stems from their metabolites that inhibit the essential enzyme acetylcholinesterase. Our goal is to develop a new treatment strategy for parathion intoxication by combining a catalytic bioscavenger that rapidly degrades the intoxicating parathion-metabolite (paraoxon) in the blood, with a glutamate bioscavenger that reduces the elevated concentration of extracellular glutamate in the brain following OP intoxication. We report on the development of a novel catalytic bioscavenger by directed evolution of serum paraoxonase 1 (PON1) that effectively detoxifies paraoxon in-vivo. We also report preliminary results regarding the utilization of this PON1 variant together with a recombinant human enzyme glutamate oxaloacetate transaminase 1 (rGOT1), suggesting that a dual PON-GOT treatment may increase survival and recovery from parathion and paraoxon intoxications.

Glutamate excitotoxicity is a primary contributor of ischemic neuronal death and other cellular components of the neurovascular unit. Several strategies have been developed against glutamate excitotoxicity, however none of them have not shown positive results in the clinical practice so far. Nowadays, the concept of blood/brain glutamate grabbing or scavenging is well recognized as a novel and attractive protective strategy to reduce the excitotoxic effect of excess extracellular glutamate that accumulates in the brain following an ischemic stroke. The main advantage of this novel therapeutic strategy is that it occurs in the blood circulation and therefore does not affect the normal brain neurophysiology, as it has been described for other drug treatments used against glutamate excitotoxicity. In this work we report all experimental data from the beginning of our studies, focused on stroke pathology, and we describe new findings about the potential application of this therapy. Future clinical trials will allow to know the real efficacy of this novel therapeutic strategy in stroke patients.

Recent studies have shown that blood glutamate grabbing is an effective strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. The purpose of the study was to investigate the effect of two of the most efficient blood glutamate grabbers (oxaloacetate and recombinant glutamate oxaloacetate transaminase 1: rGOT1) in a rat model of intracerebral hemorrhage (ICH). Intracerebral hemorrhage was produced by injecting collagenase into the basal ganglia. Three treatment groups were developed: a control group treated with saline, a group treated with oxaloacetate, and a final group treated with human rGOT1. Treatments were given 1 hour after hemorrhage. Hematoma volume (analyzed by magnetic resonance imaging (MRI)), neurologic deficit, and blood glutamate and GOT levels were quantified over a period of 14 days after surgery. The results observed showed that the treatments used induced a significant reduction of blood glutamate levels; however, they did not reduce the hematoma, nor did they improve the neurologic deficit. In the present experimental study, we have shown that this novel therapeutic strategy is not effective in case of ICH pathology. More importantly, these findings suggest that blood glutamate grabbers are a safe treatment modality that can be given in cases of suspected ischemic stroke without previous neuroimaging.

This study describes the use of in vivo magnetic resonance spectrocopy (MRS) to monitor brain glutamate and lactate levels in a paraoxon (PO) intoxication model. Our results show that the administration of recombinant glutamate-oxaloacetate transaminase (rGOT) in combination with oxaloacetate (OxAc) significantly reduces the brain-accumulated levels of glutamate. Previously we have shown that the treatment causes a rapid decrease of blood glutamate levels and creates a gradient between the brain and blood glutamate levels which leads to the efflux of excess brain glutamate into the blood stream thereby reducing its potential to cause neurological damage. The fact that this treatment significantly decreased the brain glutamate and lactate levels following PO intoxication suggests that it could become a new effective neuroprotective agent.

In the past two decades we have seen a very important advance in the research of the biology of Entamoeba histolytica. This dramatic progress has been mostly the result of (1) the introduction of a transfection system to express exogenous genes and to up- and downregulate the expression of selected genes, (2) the completion of the parasites genome sequencing, including the genomes of various Entamoeba spp. and some of its strains, and (3) the development of microarrays both for genomic and for transcriptional profiling. The introduction of these molecular tools has significantly expanded our ability to investigate many important questions such as the role and molecular mechanisms of different virulence factors, transport, and metabolic systems, as well in the cell cycle and differentiation of the parasite. In addition, it enabled the better understanding of the molecular differences among the various Entamoeba species and strains. Nevertheless, there are still many open questions that need to be answered.

Blood glutamate scavenging is a novel and attractive protecting strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. Glutamate oxaloacetate transaminase 1 (GOT1) activation by means of oxaloacetate administration has been used to reduce the glutamate concentration in the blood. However, the protective effect of the administration of the recombinant GOT1 (rGOT1) enzyme has not been yet addressed in cerebral ischemia. The aim of this study was to analyze the protective effect of an effective dose of oxaloacetate and the human rGOT1 alone and in combination with a non-effective dose of oxaloacetate in an animal model of ischemic stroke. Sixty rats were subjected to a transient middle cerebral artery occlusion (MCAO). Infarct volumes were assessed by magnetic resonance imaging (MRI) before treatment administration, and 24 h and 7 days after MCAO. Brain glutamate levels were determined by in vivo MR spectroscopy (MRS) during artery occlusion (80 min) and reperfusion (180 min). GOT activity and serum glutamate concentration were analyzed during the occlusion and reperfusion period. Somatosensory test was performed at baseline and 7 days after MCAO. The three treatments tested induced a reduction in serum and brain glutamate levels, resulting in a reduction in infarct volume and sensorimotor deficit. Protective effect of rGOT1 supplemented with oxaloacetate at 7 days persists even when treatment was delayed until at least 2 h after onset of ischemia. In conclusion, our findings indicate that the combination of human rGOT1 with low doses of oxaloacetate seems to be a successful approach for stroke treatment.

Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis.

Objective: Based on the capacity of the blood-resident enzyme glutamate oxaloacetate transaminase (GOT) to metabolize blood glutamate, our aim was to study the association of GOT activity with serum glutamate levels and clinical parameters in patients with migraine. Methods: This case-control study included 45 episodic migraine patients (IHS 2004 criteria) and 16 control subjects. We analyzed glutamate and GOT activity in peripheral blood samples obtained during interictal periods and migraine attacks (n=15). Frequency, severity, and duration of attacks and time of evolution were also recorded. Results: Migraine patients showed lower GOT activity than controls (15.22.9 vs. 18.73.8 U/l) and higher levels of glutamate (153.768.6 vs. 121.559.2 mM) (all p

Human ovarian cancer cells specifically bind the isoflavone daidzein. A chemical conjugate between daidzein and the garlic enzyme alliinase was prepared. The conjugate specifically bound to ovarian cancer cells and upon addition of the prodrug alliin, it effectively produced cytotoxic allicin molecules which killed the cancer cells. In vivo targeting and antitumor effect was confirmed by NIR and bioluminescence imaging using daidzein-alliinase-CyTE- 777 conjugates and luciferase-expressing ovarian cancer cells. Co-localization of the fluorescent conjugate with bioluminescence was observed for intraperitoneal tumors while nonconjugated alliinase did not accumulate. Biodistribution studies with Europium-labeled conjugate revealed a five fold higher uptake in tumors as compared to other tissues. Treatment of tumor bearing mice with daidzein-alliinase and alliin effectively attenuated tumor progression during the first 12 days while a 5-fold increase in bioluminescence was detected in placebo-treated animals. Autopsy revealed only small individual foci of luminescence at the site of tumor cells inoculation. Histological examination of organs and tissues did not reveal any additional foci of carcinoma or signs of toxicity. These results suggest that the targeted alliinase conjugates in the presence of alliin, generated therapeutically effective levels of allicin which were capable of suppressing tumor progression of intraperitoneal ovarian cancer in an animal model.

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

S-allyl-mercapto-captopril (CPSSA) is a conjugate of captopril with allicin, an active principle in garlic with multiple beneficial actions on metabolic syndrome abnormalities, including weight preservation, observed by the authors in fructose-induced hypertensive hyperinsulinemic rats and in Koletsky rats. The aim of the study was to examine blood pressure (BP) and glucose levels in the Cohen-Rosenthal Diabetic Hypertensive (CRDH) model as well as to follow their weight preservation. CRDH rats (n=14) were fed the sugar-rich copper-free diet essential for the development of diabetes mellitus. Two months later BP and blood glucose levels were measured. CPSSA was diluted in drinking water and administered at a final dose of 53.5 mg/kg/d (n=8). Control rats (n=6) received no drug (vehicle group). In contrast to control group, CPSSA prevented progressive weight gain, without a detectable effect on food and water intake. CPSSA was effective in attenuating systolic and diastolic BP (P

We have previously discovered a unique mechanism of epigenetic transcriptional gene silencing in the Entamoeba histolytica trophozoites of strain HM-1:IMSS that resulted in the persistent downregulation of the amoebapore A (ap-a) gene, and that could be successfully applied to silence other virulence genes (cpA5, lgl1). In order to understand how the silencing is maintained throughout generations, we analysed whether modifications occurred at the chromatin level. Chromatin immunoprecipitation assays were done with antibodies specific to the methylated lysine 4 of E. histolytica histone H3. When the genes were in a transcriptionally silent state, the methylation levels of H3K4 in their coding region were significantly reduced. In contrast, the levels of core histone H3 were consistently higher in the silenced genes. Controlled chromatin digestion with micrococcal nuclease was used to assess changes in nucleosome compaction. We found a significant resistance to digestion in the promoter region of the silenced ap-a and cpA5 genes as compared to the parental strain that expresses those genes. Our data lend further support to the idea that histone modifications and heterochromatin formations are at the basis of the transcriptional silencing of genes in E. histolytica.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The high morbidity and mortality rates as well as the poor efficacy of antifungal agents remain major clinical concerns. Allicin (diallyl-dithiosulfinate), which is produced by the garlic enzyme alliinase from the harmless substrate alliin, has been shown to have wide-range antifungal specificity. A monoclonal antibody (MAb) against A. fumigatus was produced and chemically ligated to the enzyme alliinase. The purified antibody-alliinase conjugate bound to conidia and hyphae of A. fumigatus at nanomolar concentrations. In the presence of alliin, the conjugate produced cytotoxic allicin molecules, which killed the fungus. In vivo testing of the therapeutical potential of the conjugate was carried out in immunosuppressed mice infected intranasally with conidia of A. fumigatus. Intratracheal (i.t.) instillation of the conjugate and alliin (four treatments) resulted in 80 to 85% animal survival (36 days), with almost complete fungal clearance. Repetitive intratracheal administration of the conjugate and alliin was also effective when treatments were initiated at a more advanced stage of infection (50 h). The fungi were killed specifically without causing damage to the lung tissue or overt discomfort to the animals. Intratracheal instillation of the conjugate without alliin or of the unconjugated monoclonal antibody significantly delayed the death of the infected mice, but only 20% of the animals survived. A limitation of this study is that the demonstration was achieved in a constrained setting. Other routes of drug delivery will be investigated for the treatment of pulmonary and extrapulmonary aspergillosis.

Our knowledge of the functions of different structural proteins and virulence factors in the cellular organization and pathogenesis of Entamoeba histolytica has significantly increased following the introduction of various molecular techniques that enable the manipulation of gene expression. Unfortunately, to date, all the attempts to integrate exogenous DNA into the parasite's genome have failed and most methods for up- and down-regulation of gene expression have been based on the transfection of stably maintained plasmids. Down-regulation has been achieved by plasmids encoding: (i) antisense RNA, (ii) truncated or mutated genes that exert dominant-negative effects, and (iii) inverted loops that generate double stranded RNA molecules. Small interfering RNA oligonucleotides incorporated directly from the culture medium also cause the down-regulation of specific genes. Recently, epigenetic silencing of the amoebapore gene, which encodes a toxic polypeptide that disrupts the membranes of bacteria and mammalian cells, was induced following transfections with a plasmid construct containing an upstream segment of the amoebapore gene. A number of additional genes have been silenced subsequently in a similar way. Amoebae in which the expression of virulence genes has been down-regulated have in many cases an altered phenotype that sheds light on the specific role of the genes and the cells display a significantly reduced pathogenic potential.

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

Background: Allicin in garlic is the primary active compound known to rapidly interact with free thiols. Aims of the study: To examine the effect of allicin on gene expression and glutathione cellular level in vascular endothelial cells. Methods: Cultured endothelial cells were exposed to allicin; mRNA was prepared and subjected to Micro-array and Real-Time PCR. Glutathione cellular level was determined on cell lysates. Results: Micro-array analysis demonstrated allicin-induced up- and down-regulation of 116 and 100 genes, respectively. Up-regulated genes included the phase II detoxifying enzymes thioredoxin reductase 1 and 2, heme oxygenase-1 and glutamate cysteine lygaze modifier subunit, the rate limiting enzyme in glutathione biosynthesis. Endothelial cells exposed to allicin and its derivatives containing glutathione or cysteine residues increased cellular glutathione. Allicin increased the glutathione level in a concentration and time-dependent manner up to 8-fold at a concentration of 10-20 μM after 28 h exposure. Furthermore, allicin derivative-treated cultures demonstrated a 50% decrease in tBuOOH cytotoxicity. Conclusions: These results may suggest a putative role for allicin and its derivatives in preventing reactive oxygen species damage by up-regulating the phase II detoxifying enzymes and increasing the cellular glutathione level.

Biologically active S-allylthio derivatives of 6-mercaptopurine (6-MP) and 6-mercaptopurine riboside (6-MPR) were synthesized. The products, S-allylthio-6-mercaptopurine (SA-6MP) and S-allylthio-6-mercaptopurine riboside (SA-6MPR) were characterized. The antiproliferative activity of the new prodrugs was tested on human leukemia and monolayer cell lines, and compared to that of their parent reactants. The new prodrugs acted by a concentration-dependent mechanism. They inhibited cell proliferation and induced-apoptosis more efficiently than the parent molecules. Leukemia cell lines were more sensitive to the new prodrugs than monolayer cell lines. Higher hydrophobicity of the derivatives improves their penetration into cells, where upon reaction with glutathione, S-allylthioglutathione (GSSA) is formed, and 6-MP or 6-MPR is released for further processing.

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing^R technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.

Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S-S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored-SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two -SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free -SH groups. This allowed the oriented conjugation of alliinase, via the -SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin. Published by Wiley-Blackwell.

The human intestinal pathogen Entamoeba histolytica has a number of virulence factors which can cause damage to the host. Transcriptional silencing of the gene coding for one of its major toxic molecules, the amoebapore (Ehap-a), occurred following the transfection of amoebic trophozoites with a plasmid containing the 5 promoter region of Ehap-a as well as a truncated segment of a neighboring, upstream SINE1 element that is transcribed from the opposite strand. Silencing was dependent on the presence of the truncated SINE1 sequences. Small amounts of short (∼140 n), ssRNA molecules with homology to SINE1 were detected in the silenced amoeba but no siRNA. The silenced Ehap-a gene domain had a chromatin modification indicating transcriptional inactivation without any DNA methylation. Removal of the plasmid did not restore transcription of Ehap-a. Transcription analysis by microarrays revealed that a number of additional genes were silenced and some were also up-regulated. Transfections of amoeba which already had a silenced Ehap-a, with a plasmid containing a second gene ligated to the 5 upstream region of Ehap-a, enabled the silencing, in-trans, of other genes of choice. The nonvirulent phenotype of the gene-silenced amoeba was demonstrated in various assays and the results suggest that they may have a potential use for vaccination.

The Entamoeba histolytica cell surface Ga1/Ga1NAc-inhibitable lectin is a heterodimer between a heavy (170 kDa) subunit linked via a disulfide bond to a light (31 to 35 kDa) subunit. Five light subunit genes with high homology have been identified (Ehlgl1 to -5). We have previously shown that silencing of the expression of Ehlgl1, in the G3 trophozoites which had already been silenced in the amoebapore gene (Ehap-a), also suppressed the transcription of Ehlgl1 and -3 (strain RBV). The total absence of the IgI1 to -3 subunits in the RBV trophozoites affected their ability to cap the surface Gal-lectin molecules to the uroid region. We have now found that in the RBV trophozoites, the IgI4 and -5 subunits (31 kDa) are overexpressed and appear to compensate for the missing IgI1 to -3 in the heterodimer complex. Transcriptional silencing of Ehlgl5 was achieved by transfection of G3 trophozoites with a plasmid containing the open reading frame of Ehlgl5 ligated to the 5 promoter region of the Ehap-a gene. The transfected trophozoites (strain L5) were silenced in Ehlgl5 and the closely related Ehlgl4, while the expression of the larger IgI1 to -3 subunits was upregulated. L5 trophozoites retained their ability to cap the Gal-lectin molecules. Attempts to simultaneously silence all of the Ehlgl genes have failed so far, possibly due to their crucial importance to the Gal-lectin functions. Our ability to silence part of the genes belonging to the same family can serve as a tool to study the relationships and functions of the members of other gene families.

The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LTM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.

Background: Hypertension often coexists with hyperlipidemia, insulin resistance, and glucose intolerance in metabolic syndrome. Allylmercaptocaptopril is a conjugate of the angiotensin-converting enzyme inhibitor captopril with allicin, an active principle in garlic with multiple beneficial actions on metabolic-syndrome abnormalities. We sought to test the hypothesis that the conjugation of allicin to captopril may confer additional therapeutic actions in metabolic disease. Methods: We compared allylmercaptocaptopril (53.5 mg/kg/day orally for 60 days) to an equimolar dose of captopril (40 mg/kg/day) in the spontaneously hypertensive, obese rat (SHROB) model. Results: Allylmercaptocaptopril prevented progressive weight gain, without a detectable effect on food intake. Both captopril and allylmercaptocaptopril lowered blood pressure, but allylmercaptocaptopril was more effective. Allylmercaptocaptopril, but not captopril, improved cardiac hypertrophy, as indicated by heart weight and ventricular-wall thickness. Allylmercaptocaptopril improved, whereas captopril impaired, oral glucose tolerance after a fast. Triglycerides were decreased by both captopril and allylmercaptocaptopril. Total cholesterol and non-HDL cholesterol were reduced by captopril but not by allylmercaptocaptopril. The SHROB rats developed severe glomerulosclerosis and renal failure. Allylmercaptocaptopril showed significant nephro-protection, as indicated by reductions in urinary protein loss, urinary protein-to-creatinine ratio, and plasma creatinine. Captopril showed the same trends and also prevented the decline of creatinine clearance. Finally, both allylmercaptocaptopril and captopril reduced the basal level of lipolysis in isolated abdominal adipocytes, and restored the response to catecholamine stimulation. Conclusions: Both captopril and allylmercaptocaptopril are effective in attenuating multiple abnormalities of metabolic syndrome. Allylmercaptocaptopril may have additional effectiveness on improving glucose tolerance, further lowering blood pressure, reducing cardiac hypertrophy, preventing weight gain, and protecting against renal disease.

Allicin (diallyl thiosulfinate) is a major biologically active component of garlic that is known to inhibit cell proliferation and induce apoptosis. The effects of allicin are attributed to its ability to react with thiol groups. However, the mechanism underlying the cytostatic activity of allicin, as well as the identity of the relevant subcellular targets, are not known. In the present study, we found that the effects of allicin on cell polarization, migration, and mitosis are similar to the effects of microtubule-depolymerizing drugs such as nocodazole. Moreover, treatment of cultured fibroblasts with micromolar doses of allicin results in microtubule depolymerization in cells within minutes of its application, without disrupting the actin cytoskeleton or inducing direct cytotoxic effects. Furthermore, allicin blocks the polymerization of pure tubulin in vitro in a concentration-dependent manner, suggesting that it acts directly on tubulin dimers. Sulfhydryl (SH)-reducing reagents such as 2-mercaptoethanol and dithiothreitol abolish the effect of allicin on microtubule polymerization. Thus, allicin is a potent microtubule-disrupting reagent interfering with tubulin polymerization by reaction with tubulin SH groups.

Alliinase (alliin lyase EC 4.4.1.4), a PLP-dependent α, β-eliminating lyase, constitutes one of the major protein components of garlic (Alliium sativum L.) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl-L-cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo-enzyme was crystallized in tetragonal form. These structures of alliinase with associated glycans were resolved to 1.4 Å and 1.61 Å by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to "short chain complex vacuole type" oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the ε-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo-form is nearly identical to that seen in the holo-form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.

The protozoan Entamoeba histolytica causes intestinal inflammation and liver abscess. Cysteine proteinases (CPs) have been proposed as important virulence factors for amoebiasis. To test the role of the various CPs for amoeba induced pathology, the three major enzymes of the parasite, namely EhCP1, EhCP2 and EhCP5 accounting for about 90% of total proteinase activity, were overexpressed by stable episomal transfection. Total CP activity of recombinant amoebae increased by three- to six-fold depending on the gene transfected. Interestingly, overexpression of the genes for EhCP1 or EhCP2 increased the activity of the corresponding enzyme only, whereas overexpression of the gene for EhCP5 increased the activity of all three enzymes, which is consistent with enzyme-converting activity of EhCP5. Cytopathic activity, measured by in vitro monolayer disruption, was dramatically increased in ehcp5-transfectants (five-fold) but showed only a modest increase in ehcp1- or ehcp2-transfectants (1.5-2-fold). In addition, overexpression of ehcp5 but not of ehcp1 or ehcp2 significantly increased amoebic liver abscess formation in laboratory animals. Moreover, transfection and overexpression of ehcp5 was able to compensate the reduction of in vivo pathogenicity in parasites, which have been silenced for the gene encoding the pore-forming protein amoebapore A. In summary, these results further support the important role of EhCP5 in E. histolytica pathogenicity.

Entamoeba histolytica is a protozoan parasite of humans that causes 40 000-100 000 deaths annually. Clinical amoebiasis results from the spread of the normally luminal parasite into the colon wall and beyond; the key development in understanding this complex multistage process has been the publication of the E. histolytica genome, from which has come an explosion in the use of microarrays to examine changes in gene expression that result from changes in growth conditions. The genome has also revealed a unique arrangement of tRNA genes and an extraordinary number of genes for putative virulence factors, many unexpressed under the artificial conditions of growth in culture. The ability to induce apoptosis of mammalian cells and a useful, but as yet little-understood, technique for epigenetic irreversible gene silencing are other exciting developments.

The incidence of malaria is increasing, and there is an urgent need to identify new drug targets for both prophylaxis and chemotherapy. Potential new drug targets include Plasmodium proteases that play critical roles in the parasite life cycle. We have previously shown that the major surface protein of Plasmodium sporozoites, the circumsporozoite protein (CSP), is proteolytically processed by a parasite-derived cysteine protease, and this processing event is temporally associated with sporozoite invasion of host cells. E-64, a cysteine protease inhibitor, inhibits CSP processing and prevents invasion of host cells in vitro and in vivo. Here we tested allicin, a cysteine protease inhibitor found in garlic extracts, for its ability to inhibit malaria infection. At low concentrations, allicin was not toxic to either sporozoites or mammalian cells. At these concentrations, allicin inhibited CSP processing and prevented sporozoite invasion of host cells in vitro. In vivo, mice injected with allicin had decreased Plasmodium infections compared to controls. When sporozoites were treated with allicin before injection into mice, malaria infection was completely prevented. We also tested allicin on erythrocytic stages and found that a 4-day regimen of allicin administered either orally or intravenously significantly decreased parasitemias and increased the survival of infected mice by 10 days. Together, these experiments demonstrate that the same cysteine protease inhibitor can target two different life cycle stages in the vertebrate host.

In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5 upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5 upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development.

Gene expression analysis by microarray assay revealed that when exposed to stress, Entamoeba histolytica exhibits a specific heat shock response, together with a dramatic overall reduction in gene transcription as well as differential allelic expression of key genes participating in virulence, such as the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin.

Objective: Garlic (Allium sativum) has been suggested to affect several cardiovascular risk factors. Its antiatherosclerotic properties are mainly attributed to allicin that is produced upon crushing of the garlic clove. Most previous studies used various garlic preparations in which allicin levels were not well defined. In the present study, we evaluated the effects of pure allicin on atherogenesis in experimental mouse models. Methods and Results: Daily dietary supplement of allicin, 9 mg/kg body weight, reduced the atherosclerotic plaque area by 68.9 and 56.8% in apolipoprotein E-deficient and low-density lipoprotein (LDL) receptor knockout mice, respectively, as compared with control mice. LDL isolated from allicin-treated groups was more resistant to CuSO ₄-induced oxidation ex vivo than LDL isolated from control mice. Incubation of mouse plasma with ³H-labeled allicin showed binding of allicin to lipoproteins. By using electron spin resonance, we demonstrated reduced Cu ²⁺ binding to LDL following allicin treatment. LDL treatment with allicin significantly inhibited both native LDL and oxidized LDL degradation by isolated mouse macrophages. Conclusions: By using a pure allicin preparation, we were able to show that allicin may affect atherosclerosis not only by acting as an antioxidant, but also by other mechanisms, such as lipoprotein modification and inhibition of LDL uptake and degradation by macrophages.

Background & Aims: The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. Methods: Cysteine protease-deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [ ³⁵S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. Results: Trophozoites with reduced cysteine protease activity were ineffective at degrading [³⁵S] cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease-deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. Conclusions: These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis.

Transcriptional silencing of an amebapore (ap-a) gene occurred in Entamoeba histolytica following the transfection of plasmids containing a DNA segment (473 bp) homologous to the 5 upstream region of the gene. This segment contains the promoter region of the ap-a gene, a T-rich stretch, followed by a truncated SINE1 (short interspersed element) that is transcribed from the opposite strand. The downstream silencing of the ap-a gene did not occur with plasmids containing the entire SINE1 sequence or lacking the entire SINE1 sequences including the T-rich stretch. Such plasmids promoted the overexpression of the ap-a gene. The transcription of the SINE element required both the T-rich stretch as well as sequences from the 5 end of SINE. RNA extracts from gene-silenced cultures showed small amounts of short (∼140 nt), single-stranded molecules with homology to SINE1 transcripts but no siRNA. Chromatin immunoprecipitation (ChIP) analysis of silenced G3 trophozoites with an antibody against methylated K4 of histone H3 revealed a demethylation of K4 at the domain of the ap-a gene indicating transcriptional inactivation. These results suggest the involvement of the SINE1 element in triggering the gene silencing and the role of histone modification in its epigenetic maintenance. The avirulent phenotype of the silenced trophozoites was demonstrated in various assays and the results suggest they may have a potential use for vaccination.

Pure allicin, prepared biosynthetically by reacting synthetic alliin with an immobilized alliinase enzyme, is known to possess cardioprotective effects. However, in its pure form, allicin is pharmacologically unstable. S-allylmercaptocaptopril (CPSSA) is a new stable synthetic compound produced by chemical reaction between allicin and the angiotensin converting enzyme inhibitor captopril. Using the fructose-induced metabolic syndrome rat model we studied the effects of short-term treatment with two doses of CPSSA on cardiovascular risk factors associated with the metabolic syndrome, in comparison to the effects of allicin and captopril separately. Allicin (8 mg/(kg day)) significantly reduced insulin, triglycerides, and homocysteine concentrations, and had a slight effect on SBP. Captopril (50 mg/(kg day)) only improved blood pressure and homocysteine. Treatment with low dose of CPSSA (5 mg/(kg day)) lowered SBP but did not improve any other measured parameter, while treatment with a higher dose (50 mg/(kg day)) significantly decreased blood pressure, triglycerides, and homocysteine concentrations. We conclude that the combined molecule CPSSA integrates the anti-hypertensive, lipid-lowering, and homocysteine-reducing effects of both allicin and captopril, making it a potential cardiovascular protective agent.

Transcriptional silencing of an amoebapore (ap-a) gene occurred in Entamoeba histolytica following the transfection of plasmids containing a DNA segment (473 bp) homologous to the 5 upstream region of the gene (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). This segment contains the promoter region of the ap-a gene, a T-rich stretch, followed by a truncated SINE1 (short interspersed element 1) that is transcribed from the antisense strand. Transfection of plasmids containing truncated SINE1 sequences which lack their 3 regulatory elements upstream of the ap-a gene was essential for the downstream silencing of the ap-a gene while transfection with plasmids containing the entire SINE1 sequence or without the T-rich stretch promoted the overexpression of the ap-a gene. Both the T-rich stretch and sequences of the 5 SINE1 were essential for the transcription of SINE1. RNA extracts from gene-silenced cultures showed small amounts of short (∼140-nucleotide), single-stranded molecules with homology to SINE1 but no short interfering RNA. Chromatin immunoprecipitation analysis with an antibody against methylated K4 of histone H3 showed a demethylation of K4 at the domain of the ap-a gene, indicating transcriptional inactivation. These results suggest the involvement of SINE1 in triggering the gene silencing and the role of histone modification in its epigenetic maintenance.

Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37°C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.

Entamoeba histolytica is a phagocytic cell with numerous vesicles of different sizes and shapes but without a well-defined Golgi apparatus. Despite this, genes implied in membrane trafficking have been identified in the genome of this parasite. One of these genes is homologous to the N-ethylmaleimide sensitive fusion factor (NSF), whose protein has been shown to play an important role in vesicle fusion in other eukaryotic cells. In this report, we investigated the NSF homologue gene from a pathogenic E. histolytica, characterized its protein product and two of its activities, ATPase and in vitro intra-Golgi transport. The finding of an active NSF protein in E. histolytica indicates that a simple or primordial Golgi apparatus probably exists in this microorganism, as has been proposed by others.

In cells, the α-anomers of aldoses are the preferred metabolizable substrates, while β-anomers of aldoses play their role in glycan structure. In the cytoplasm, α- and β-anomers of aldoses interconvert through the enzyme termed aldose 1-epimerase or mutarotase (EC 5.1.3.3). We have identified a mutarotase gene in Entamoeba histolytica, the causative agent of non-bacterial dysentery in humans. Cloning and characterization of this gene in two strains of the parasite (HM-1:IMSS and Rahman) that differ in their pathogenicity, revealed that the sequence is identical in both strains. A recombinant E. histolytica mutarotase was produced as well as specific antibodies that recognized a 38 kDa protein in trophozoite lysates of both strains. Mutarotase activity was observed with the recombinant protein as well as in lysates of both HM-1:IMSS and Rahman, the former exhibiting a slightly higher mutarotase activity. Finally, we have shown by complementation that overexpression of the E. histolytica mutarotase in a mutarotase defective Escherichia coli strain restores the ability of these bacteria to grow in minimal medium with phenyl-β-galactopyranoside as the sole carbon source.

Allicin, a highly active component from freshly crushed garlic, is produced upon the reaction of the small molecular weight molecule alliin, with the enzyme alliinase (EC 4.4.1.4). Because allicin was shown to be toxic to various mammalian cells in vitro, we devised a novel approach for the therapy of B-cell malignancies based on site-directed generation of allicin. Alliinase was conjugated to the monoclonal antibody rituximab, which recognizes the CD20 antigen, and the resulting conjugate was targeted to CD20⁺ B chronic lymphocytic leukemia (B-CLL) and other B-cell lymphomas. Upon addition of alliin, allicin was formed in situ, killing the CD20⁺ tumor B cells via apoptosis. Following a 72-hour treatment, an 85% and 96% reduction was observed in the number of viable B-CLL and EBV-transformed B cells, respectively. Using the human/mouse radiation chimera for the evaluation of allicin targeting in a preclinical animal model, we showed a significant reduction in the number of recovered B-CLL, mantle cell lymphoma, or EBV-transformed B cells. We conclude that our system offers a new powerful and less toxic therapy for B-CLL and other B-cell malignancies. Furthermore, combining alliinase with the appropriate monoclonal antibody may extend the application of this approach to other conditions in which the elimination of a specific cell population is desired.

Allicin (diallylthiosulfinate), the active substance of garlic, has been shown to possess a variety of biological activities. Mechanistic and pharmacokinetic studies of allicin and its derivatives raise the need for a labeled compound. However, labeling of this volatile and unstable liquid requires delicate handling. Here, we describe a simple method for the preparation of ³H-labeled allicin. This was achieved by applying synthetic [³H]alliin ([2,3-³H]allylcysteine sulfoxide) to a column containing immobilized alliinase [EC 4.1.1.4.] from garlic. Purification of [³H]allicin was done by differential adsorbtion of the reaction components on a neutral polystyrene resin, Porapak Q. Thiol-containing compounds are known to be the main target of allicin. In this work we demonstrated that [³H]allicin can be used for the synthesis of labeled [³H]allylmercapto derivatives of SH peptides and proteins. Thus, we prepared [³H]S-allylmercaptoglutathione which can be used in metabolic studies. Moreover, we showed that incubation of alliinase with [³H]allicin led to modification of 1.4 cysteine residues per subunit of the enzyme.

Objectives: The evaluation of allicin, the biologically active compound responsible for the antimicrobial activities of freshly crushed garlic cloves, in inhibiting Aspergillus spp. in vitro and in a murine model of disseminated aspergillosis. Methods: Pure allicin was prepared by reacting synthetic alliin with a stabilized preparation of the garlic enzyme alliinase. We tested the in vitro efficacy of pure allicin against 31 clinical isolates of Aspergillus spp. using a microdilution broth method and following the NCCLS guidelines (document M-38P). Subsequently, the in vivo efficacy of allicin was tested in immunocompetent mice infected intravenously (iv) with Aspergillus fumigatus conidia. Allicin (5 mg/kg body weight) was administered iv once daily for 5 days post-infection or orally (po) (9 mg/kg body weight) for 5 days pre-infection and 10 days post-infection. No ill effects were observed in allicin-treated uninfected mice. Results: The in vitro MICs and MFCs of allicin were between 8 and 32 mg/L, indicating that allicin in its pure form may be an effective fungicide in vitro. Time-kill studies indicate that allicin exerts its fungicidal activity within 2-12 h of administration in vitro. Allicin treatment significantly prolonged survival of infected mice (P < 0.01) from mean survival time (MST) = 7.7 days in untreated mice to MST = 21.3 and 13.9 days for allicin iv and po treated mice, respectively. Allicin iv treatment led to a significant (P < 0.001) 10-fold reduction in fungal burden in A. fumigatus infected mice as evaluated by quantitative fungal cultures of kidney tissue samples. Conclusions: These favourable results, despite the short half-life of this compound in vivo, support further studies of controlled sustained release or more prolonged administration of allicin as a treatment for aspergillosis.

Allicin, a major ingredient of fresh garlic extract that is produced during the crushing of garlic cloves, exerts various beneficial biological effects, including a broad spectrum of antimicrobial activity, antihyperlipidaemic and antihypertensive effects. However, how allicin affects the immune system is less well known, and its effect on human T cells has never been studied. Here, we examined the in-vitro effects of allicin on the functioning of T cells related to their entry to inflamed extravascular sites. We found that allicin (20-100 μM) inhibits the SDF-1α (CXCL12)-induced T cell migration through fibronectin (FN), and that this inhibition is mediated by the down-regulation of (i) the reorganization of cortical actin and the subsequent T cell polarization, and (ii) T cell adhesion to FN. Moreover, allicin also inhibited T cell adhesion to endothelial cells and transendothelial migration. The mechanisms underlying these inhibitory effects of allicin are associated with its ability to down-regulate the phosphorylation of Pyk2, an intracellular member of the focal adhesion kinases, and to reduce the expression of the VCAM-1- and FN-speciflc α4β1-integrin (VLA-4). The ability of allicin to down-regulate these chemokine-induced and VLA-4-mediated T cell functions explains its beneficial biological effects in processes where T cells play an important role and suggests that allicin may be used therapeutically with chronic inflammatory diseases.

Allicin, the main organic allyl sulfur component in garlic, exhibits immune-stimulatory and antitumor properties. Allicin stimulated [³H]thymidine incorporation in mouse splenocytes and enhanced cell-mediated cytotoxicity in human peripheral mononuclear cells. Multiple administration (i.p.) of allicin elicited a marked antitumor effect in mice inoculated with B-16 melanoma and MCA-105 fibrosarcoma. The immune-stimulatory and antitumor effects of allicin are characterized by a bell-shaped curve, i.e. allicin at high, supra-optimal concentrations is less effective or inhibitory. Allicin induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in human peripheral mononuclear cells, and also in wild-type Jurkat T-cells. Allicin failed to activate ERK1/2 in Jurkat T cells that express p21^ras, in which Cys118 was replaced by Ser. These cells are not susceptible to redox-stress modification and activation. We postulate that the immune stimulatory effect of allicin is mediated by redox-sensitive signaling such as activation of p21^ras. It is suggested that the antitumor effect of allicin is related to its immune-stimulatory properties.

Entamoeba histolytica trophozoites produce amoebapores, a family of small amphipathic peptides capable of insertion into bacterial or eukaryotic membranes and causing cellular lysis. Recently, E. histolytica trophozoites that are totally deficient in the production of amoebapore-A were created through a gene silencing mechanism (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). Here we tested the virulence of amoebapore A(-) trophozoites in models of the two major forms of amebic disease: amebic liver abscess and amebic colitis. We demonstrate that amoebapore expression is required for full virulence in the SCID mouse model of amebic liver abscess, but E. histolytica trophozoites that do not express amoebapore-A can still cause inflammation and tissue damage in infected human colonic xenografts. These data are consistent with the concept that tissue damage may proceed by different mechanisms in amebic liver abscess compared to amebic colitis.

Allylmercaptocaptopril (CPSSA) was synthesized by reacting captopril with pure allicin. Fructose-induced hypertensive groups of rats were fed a fructose-rich diet for 3 weeks, and then received the diet plus either CPSSA (40 to 56 mg or 138 to 194 μmol/L/kg/d) or captopril (80 mg or 369 μmol/L/kg/d) for 2 more weeks. CPSSA (both doses) significantly lowered blood pressure (BP) from 153.4 to 120.8 mm Hg (P < .005). Captopril gave similar results, lowering BP from 150.7 to 123 mm Hg (P < .005). CPSSA also decreased the high levels of triglycerides to normal. The new stable compound allylmercaptocaptopril combines the beneficial properties of captopril and allicin and is a potential candidate for antihypertensive drug therapy.

We investigate the final stage of cytokinesis in two types of amoeba, pointing out the existence of biphasic furrow contraction. The first phase is characterized by a constant contraction rate, is better studied, and seems universal to a large extent. The second phase is more diverse. In Dictyostelium discoideum the transition involves a change in the rate of contraction, and occurs when the width of the cleavage furrow is comparable to the height of the cell. In Entamoeba invadens the contractile ring carries the cell through the first phase, but cannot complete the second stage of cytokinesis. As a result, a cooperative mechanism has evolved in that organism, where a neighboring amoeba performs directed motion towards the dividing cell, and physically causes separation by means of extending a pseudopod. We expand here on a previous report of this novel chemotactic signaling mechanism.

Entamoeba histolytica, the protozoan parasite which causes amoebiasis, is an exclusively human pathogen so developing a vaccine could effectively impact the spread of the disease. Recently we developed a genetically modified avirulent strain, termed G3, from the virulent E. histolytica strain HM-1:IMSS. The new strain lacks the important virulence factor, the amoebapore-A. The objective of our current study was to investigate the avirulence of the attenuated strain as well as to examine the antigenic and immunogenic responses of these trophozoites as potential candidates for a live vaccine. Functional assays were conducted to characterise the virulent behaviour of the G3 strain. This behaviour was compared to the virulent strain HM-1:IMSS and the non-virulent strain Rahman. Western blots were conducted to confirm the lack of amoebapore-A in the E. histolytica G3 strain and to demonstrate that it had no influence on the presence of other virulence factors. Results of these two sets of tests proved the G3 strain to be phenotypically similar to the avirulent Rahman strain while antigenically identical to the virulent HM-1:IMSS, apart from the lack of the amoebapore-A protein. Intraperitoneal immunisation of hamsters with G3 trophozoites compared to sham immunised hamsters resulted in IgG anti-HM-1:IMSS antibodies. The level of humoral response was variable and further testing has to take place before introducing this new strain as a vaccine.

Background: Commercially available garlic preparations in the form of garlic oil, garlic powder and pills are widely used for certain therapeutic purposes, including lowering blood pressure and improving lipid profile. Despite the impressive effects of garlic most studies are limited by lack of controlled methods and suitable double-blinding, and by the use of preparations with unknown amounts and chemical identification of the active ingredient. Allicin, a synthetic preparation of an active constituent of garlic, was found to lower blood pressure, insulin, and triglycerides levels in fructose-fed rats. Thus, it was considered important to assess its effect on the weight of the animals. Methods: Male Sprague-Dawley rats weighing 240 to 250 g were fed a fructose-enriched diet consisting of 21% protein, 5% fat, 60% carbohydrate, 0.49% sodium and 0.49% potassium for 5 weeks, which produced hyperinsulinemia, hypertension, and hypertriglyceridemia. Group I (controls) rats were fed a diet enriched by fructose only; group II was given allicin the last 2 weeks, and group III was given allicin the first 3 weeks. The three groups consumed the same amount of food. Weight was measured at the beginning of the experiment and after 3 and 5 weeks on the diet. Results: Weight in the control group rose from 249.4 ± 10.04 g to 274.5 ± 15.5 g after 3 weeks and to 306.9 ± 22.2 g after 5 weeks. Weight in group II rose from baseline 259.1 ± 12.1 g to 306.9 ± 22.2 g after 3 weeks on fructose alone, and declined slightly to 282.4 ± 17.4 g after 2 weeks of allicin (P < .02). In group III, in which the protocol was reversed, the baseline weight of 260.4 ± 13.25 g rose only to 269.8 ± 15.3 g (P < .431) after 3 weeks on fructose and allicin. Conclusions: The control group that was fed a diet enriched by fructose alone continued to gain weight, whereas the groups fed allicin did not. The difficulty of preventing weight gain after reaching the nadir of weight loss underscores the practical value of allicin for weight control.

Allicin (diallyl thiosulfinate), a highly active component in extracts of freshly crushed garlic, is the interaction product of non-protein amino acid alliin (S-allyl-L-cysteine sulfoxide) with the enzyme alliinase (alliin lyase; EC 4.4.1.4). Allicin was shown to be toxic in various mammalian cells in a dose-dependent manner in vitro. We made use of this cytotoxicity to develop a novel approach to cancer treatment, based on site-directed generation of allicin. Alliinase from garlic was chemically conjugated to a mAb directed against a specific tumor marker, ErbB2. After the mAb-alliinase conjugate was bound to target tumor cells, the substrate, alliin, was added. In the presence of alliin, tumor-localized alliinase produced allicin, which effectively killed N87 and CB2, both ErbB2-expressing cells in vitro, whereas 32D cells (a murine hematopoietic progenitor cell line, devoid of the ErbB2 receptors) were not affected. Moreover, using N87, a human tumor cell line xenograft in athymic nude mice, we demonstrated for the first time, a high antitumor activity of allicin that was produced in situ by the conjugate, on alliin administration in vivo, while at the same time other tissues were unharmed due to the inert nature of alliin and the high clearance rate of allicin. The effect of the treatment on tumor growth arrest became significant 2 weeks after its onset, and it continued to rise, reaching highly significant inhibition a week later. Ten days after the end of the treatment (day 18), tumor growth inhibition was still the same.

Invasive microorganisms efface enteric microvilli to establish intimate contact with the apical surface of enterocytes. To understand the molecular basis of this effacement in amebic colitis, we seeded Entamoeba histolytica trophozoites on top of differentiated human Caco-2 cell layers. Western blots of detergent lysates from such cocultures showed proteolysis of the actin-bundling protein villin within 1 min of direct contact of living trophozoites with enterocytes. Mixtures of separately prepared lysates excluded detergent colysis as the cause of villin proteolysis. Caspases were not responsible as evidenced by the lack of degradation of specific substrates and the failure of a specific caspase inhibitor to prevent villin proteolysis. A crucial role for amebic cysteine proteinases was shown by prevention of villin proteolysis and associated microvillar alterations through the treatment of trophozoites before coculture with synthetic inhibitors that completely blocked amebic cysteine proteinase activity on zymograms. Moreover, trophozoites of amebic strains pSA8 and SAW760 with strongly reduced cysteine proteinase activity showed a reduced proteolysis of villin in coculture with enteric cells. Salmonella typhimurium and enteropathogenic Escherichia coli disturb microvilli without villin proteolysis, indicating that the latter is not a consequence of the disturbance of microvilli. In conclusion, villin proteolysis is an early event in the molecular cross-talk between enterocytes and amebic trophozoites, causing a disturbance of microvilli.

Transcriptional silencing of the gene coding for amoebapore A (AP-A) was observed when trophozoites of Entamoeba histolytica were transfected with a hybrid plasmid construct containing the ap-a gene flanked by the upstream and downstream segments of the original Ehap-a gene. Transfectants were totally devoid of ap-a transcript and AP-A protein. An identical silencing effect was observed upon transfection with a plasmid that contained only the 5 upstream region of ap-a. Removal of the selecting antibiotic enabled the isolation of plasmidless clones, which retained in their progeny the silenced phenotype. E. histolytica cells were able to overexpress ap-a when transfected with a plasmid containing the gene flanked by the 5 and 3 regions of the EhRP-L21 gene. This plasmid, however, could not express ap-a in the retransfected, cloned trophozoites lacking AP-A. This is the first report of gene silencing in E. histolytica, and the mechanism appears to belong to transcriptional gene silencing and not to posttranscriptional gene silencing. This conclusion is based on the following results: (i) silencing was achieved by transfection of homologous 5 flanking sequences (470 bp of the Ehap-a gene), (ii) transcription initiation of Ehap-a was found to be blocked, and (iii) short double-stranded RNA fragments of the ap-a coding and noncoding sequences were not detected. Trophozoites lacking AP-A are nonpathogenic and impaired in their bacteriolytic capability.

The 260-kDa heterodimeric Gal/GalNAc-specific Lectin (Gal-lectin) of Entamoeba histolytica dissociates under reducing conditions into a heavy (hgl, 170 kDa) and a light subunit (lgl, 35 kDa). We have previously shown that inhibition of expression of the 35-kDa subunit by antisense RNA causes a decrease in virulence. To further understand the role of the light subunit of the Gal-lectin in pathogenesis, amoebae were transfected with plasmids encoding intact, mutated, and truncated forms of the light subunit lgl1 gene. A transfectant in which the 55 N-terminal amino acids of the lgl were removed, overproduced an N-truncated lgl protein (32 kDa), which replaced most of the native 35-kDa lgl in the formation of the Gal-lectin heterodimeric complex and exerted a dominant negative effect. Amoebae transfected with this construct showed a significant decrease in their ability to adhere to and kill mammalian cells as well as in their capacity to form rosettes with and to phagocytose erythrocytes. In addition, immunofluorescence confocal microscopy of this transfectant with anti-Gal-lectin antibodies showed an impaired ability to cap. These results indicate that the light subunit has a role in enabling the clustering of Gal-lectin complexes and that its N-truncation affects this function, which is required for virulence.

We have previously demonstrated that inhibition of expression of amoebapore A (AP-A) by antisense RNA caused a marked decrease in the virulence of the parasite. A four-fold over-expression of AP-A was obtained with plasmid (pA7) which has the ap-a gene under the control of gene EhgLE-3-RP-L21. The virulence of the transfected trophozoites, however, was also decreased. Excess of AP-A protein was found in the cytosol and a significant amount was released into the surrounding media. Transfection of the parasite with a plasmid (psAP-1) in which the ap-a gene was introduced with its own regulatory sequences, caused a total suppression of the transcription and translation of both the genomic and episomal ap-a genes. The silenced transfectant was not virulent at all. These results demonstrate that important factors need to be expressed at the correct cellular location and that the parasite has additional internal control mechanisms such as transcriptional gene silencing which can prevent excess amounts of gene expression.

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (λ_max = 324 nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.

Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925 are two of eight species of Entamoeba that sometimes inhabit the human colon. The former is an invasive organism capable of causing life-threatening intestinal and extra-intestinal disease; the latter appears not to be invasive. Because the two species, when viewed by light microscopy appear morphologically similar, they were long regarded as a single species. However, recent biochemical, immunological, and genetic studies provided convincing evidence that they belong to separate species. Our ultrastructural studies revealed distinct differences in at least two features of the trophozoites. 1) The cell surfaces of the trophozoites of each species differ with regard to structures exposed on the surface, and the distribution and arrangement of intra-membranous proteins. 2) The phagocytosis of bacteria differs in respect to the formation of the phagocytic vacuoles. Loose vacuoles containing several bacteria were seen in E. histolytica whereas tight vacuoles containing a single bacterium were observed in E. dispar. Furthermore, bacteria were found only within vacuoles in E. histolytica; in E. dispar, bacteria were found within vacuoles and some were found free in the cytoplasm.

The enzyme alliinase has been isolated from garlic bulbs and crystallized. The crystals belong to space group P2₁, with unit-cell parameters a = 70.191, b = 127.006, c = 108.085 Å, β = 93.384°. They diffract to 2.2 Å at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function suggests that the crystals contain two dimeric molecules per asymmetric unit.

Asexual cells are normally able to reproduce entirely by themselves. But we have discovered that in about one-third of the dividing cells of Entamoeba invadens contraction of the cleavage furrow1 may stop before separation is complete. We show here that the connected daughter cells overcome this problem by calling upon a neighbouring amoeba to help them achieve the final stage of division. The 'midwife' cell is chemotactically recruited for this mechanical intervention in what is a surprising example of primitive cooperation.

The effects of a synthetic preparation of an active constituent of garlic, allicin, were studied on blood pressure (BP), triglycerides, and insulin levels in Sprague-Dawley rats in which high fructose feeding elicited hyperinsulinemia, hypertension, and hypertriglyceridemia. Results were compared with those of the antihypertensive drug enalapril. Three groups of male Sprague-Dawley rats were fed a fructose-enriched diet for 5 weeks. During the last 2 weeks 10 animals received only fructose, 10 received allicin, and 10 received enalapril. Blood pressure, insulin level, and triglyceride levels were measured at the beginning of the experiment and after 3 and 5 weeks on the fructose diet, fructose/allicin diet, or fructose/enalapril diet. Allicin lowered BP from the maximal level (after 3 weeks of fructose) of 153.4 ± 8 mm Hg to 139.7 ± 12 mm Hg after 2 weeks on allicin; insulin from 11.7 ± 3.7 ng/mL on fructose diet to 6.92 ± 3.3 ng/mL on allicin; and triglycerides from 132.8 ± 18 mg/dL on fructose to 59.6 ± 27 mg/dL on allicin. The similar effect of allicin and enalapril on BP, insulin, and triglycerides reinforces the trend toward combining the nonpharmacologic approach with drug therapy.

The reaction between allicin (diallylthiosulfinate), the active component of garlic and reduced glutathione was investigated. The product of this reaction, mixed disulfide S-allylmercaptoglutathione (GSSA) was separated by high performance liquid chromatography and identified by ¹H and ¹³C nuclear magnetic resonance and mass spectroscopy. The reaction is fast (with an apparent bimolecular reaction rate constant of 3.0 M^-1 s^-1). It is pH-dependent, which reveals a direct correlation to the actual concentration of mercaptide ion (GS^-). Both GSSA and S-allylmercaptocysteine (prepared from allicin and cysteine) reacted with SH-containing enzymes, papain and alcohol dehydrogenase from Thermoanaerobium brockii yielding the corresponding S-allylmercapto proteins, and caused inactivation of the enzymes. The activity was restored with dithiothreitol or 2-mercaptoethanol. In addition, GSSA also exhibited high antioxidant properties. It showed significant inhibition of the reaction between OH radicals and the spin trap 5,5'-dimethyl-1-pyroline N-oxide in the Fenton system as well as in the UV photolysis of H₂O₂. In ex vivo experiments done with fetal brain slices under iron-induced oxidative stress, GSSA significantly lowered the production levels of lipid peroxides. The similar activity of GSSA and allicin as SH-modifiers and antioxidants suggests that the thioallyl moiety has a key role in the biological activity of allicin and its derivatives. (C) 2000 Elsevier Science B.V.

Trophozoites of the parasitic protozoa, Entamoeba histolytica, synthesize a cell surface lipoglycoconjugate, termed lipophosphoglycan, which is thought to be an important virulence factor and potential vaccine candidate against invasive amebiasis. Here, we show that the E. histolytica lipophosphoglycans are in fact glycosylphosphatidylinositol (GPI)-anchored proteophosphoglycans (PPGs). These PPGs contain a highly acidic polypeptide component which is rich in Asp, Glu and phosphoserine residues. This polypeptide component is extensively modified with linear glycan chains having the general structure, [Glcα1-6](n)Glcβ1-6Gal (where n = 2-23). These glycan chains can be released after mild-acid hydrolysis with trifluoroacetic or hydrofluoric acid and are probably attached to phosphoserine residues in the polypeptide backbone. The PPGs are further modified with a GPI anchor which differs from all other eukaryotic GPI anchors so far characterized in containing a glycan core with the structure, Gal₁Man₂GlcN-myo-inositol, and in being heterogeneously modified with chains of α-galactose. Trophozoites of the pathogenic HM-1:IMSS strain synthesize two distinct classes of PPG which have polydisperse molecular masses of 50-180 kDa (PPG-1) and 35-60 kDa (PPG-2) and are modified with glucan side-chains of different average lengths. In contrast, the non-pathogenic Rahman strain synthesizes one class of PPG which is only elaborated with short disaccharide side-chains (i.e. Glcβ1-6Gal). However, the PPGs are abundant in all strains (8 x 10⁷ copies per cell) and are likely to form a protective surface coat. (C) 2000 Academic Press.

Down regulation of gene expression by antisense RNA is one of the ways to investigate the specific contribution of certain components to the physiology and activities of a cell. A successful inhibition of gene expression in Entamoeba trophozoites was achieved in stable transfectants by using hybrid plasmid constructs containing promotors that produce transcripts which do not bind to polysomes. Different promotors were found to be required for Entamoeba histolytica or Entamoeba dispar. In E. histolytica one of the two copies (g34) of the gene coding for ribosomal protein L21 was previously found to be transcribed but not translated. Inhibition of gene expression was obtained by placing in a transfection vector, the amoebapore A gene, in its antisense orientation, under the control of the g34 promotor. Transfectants of E. histolytica were shown to accumulate antisense transcripts and inhibit amoebapore synthesis. In contrast, transfectants with plasmid constructs in which the amoebapore gene was placed under the control of the gLE3 promotor of RP-L21, which is known to be translated, did not accumulate antisense transcript or inhibit gene expression. Maximal inhibition of amoebapore expression was obtained when the antisense construct also included the 5' and 3' untranslated regions of the amoebapore gene. In E. dispar the opposite situation was found, plasmid constructs containing the promotor regions of the gLE3 copy, which were shown to be poorly translated, were more efficient in inhibiting the synthesis of a 30 kDa surface-specific antigen than a construct with the g34 promotor element. (C) 2000 Elsevier Science B.V.

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols. Copyright (C) 2000 Elsevier Science B.V.

The diverse health benefit effects of garlic include its anticancer activity. However, very little is known about such activity of isolated garlic compounds, among which allicin (the major ingredient of crushed garlic) has been the least studied. The aim of this work was to determine whether pure allicin exhibits the antiproliferative effect reported for garlic in in vitro models. Allicin, but not its precursor alliin, inhibited proliferation of human mammary (MCF-7), endometrial (Ishikawa), and colon (HT-29) cancer cells (50% inhibitory concentration = 10-25 μM). Two of three tested primary lines of human fibroblasts displayed a similar response to allicin (50% inhibitory concentration = 16-40 μM), whereas the third line was almost unaffected by this compound. The pure allicin and water extract of garlic powder with equivalent allicin concentrations displayed a similar potency, suggesting that allicin is responsible for the antiproliferative effect of the extract. The growth inhibition was accompanied by accumulation of cells in the G₀/G₁ and G₂/M phases of the cell cycle (MCF-7 cells) and not by a significant increase in cell death. Allicin caused a transient drop in the intracellular glutathione (GSH) level, the magnitude and kinetics of which significantly varied depending on cell type. The extent of the decrease in GSH levels correlated well (r = O.75) with the growth inhibitory activity of allicin. On the basis of these findings, we suggest that allicin plays a major role in the antiproliferative effect of water-soluble garlic preparations and that this effect may be attributed to the ability of allicin to transiently deplete the intracellular GSH level.

Keywords: EXPRESSION

Keywords: ADHERENCE LECTIN

Keywords: CYSTEINE PROTEINASES; ANTISENSE INHIBITION; EXPRESSION

Keywords: ANTISENSE INHIBITION; GAL/GALNAC LECTIN; EXPRESSION; DECREASE; SUBUNIT

The protozoan parasite Entamoeba histolytica causes intestinal inflammation and ulceration. Amoebic trophozoites activate the transcription factor NF-κB in human intestinal epithelial cells, initiating an inflammatory response programme with resultant damage to the intestinal tissue. Amoebic cysteine proteinases have been proposed as important virulence factors for amoebiasis. To test the role of amoebic cysteine proteinases in the pathogenesis of amoebic colitis, human intestinal xenografts in SCID mice were infected with E. histolytica trophozoites expressing an antisense message to ehcp5. The cysteine proteinase-deficient amoeba failed to induce intestinal epithelial cell production of the inflammatory cytokines interleukin (IL)-1B and IL-8, and caused significantly less gut inflammation and damage to the intestinal permeability barrier. The critical role of amoebic cysteine proteinases in human gut inflammation and tissue damage may be explained by our discovery that amoebic cysteine proteinases possess IL-1B converting enzyme (ICE) activity. This ICE activity could contribute to intestinal inflammation by activating human p1L-1B released by damaged intestinal cells. These results demonstrate for the first time that amoebic cysteine proteinases are a key virulence factor in amoebic colitis, and provide a novel mechanism for their activity.

Entamoeba histolytica virulence is related to a number of amebic components (lectins, cysteine proteinases, and amebapore) and host factors, such as intestinal bacterial flora. Trophozoites are selective in their interactions with bacteria, and the parasite recognition of glycoconjugates plays an important role in amebic virulence. Long-term monoxenic cultivation of pathogenic E. histolytica trophozoites, strains HK-9 or HM-1:IMSS, with Escherichia coli serotype O55, which binds strongly to the Gal/GalNAc amebic lectin, markedly reduced the trophozoites' adherence and cytopathic activity on cell monolayers of baby hamster kidney (BHK) cells. Specific probes prepared from E. histolytica lectin genes as well as antibodies directed against the light (35-kDa) and heavy (170-kDa) subunits of the Gal/GalNAc lectin revealed a decrease in the transcription and expression of the light subunit in trophozoites grown monoxenically with E. coli O55. This effect was not observed when E. histolytica was grown with E. coli 346, a mannose- binding type I pilated bacteria. Our results suggest that the light subunit of the amebic lectin is involved in the modulation of parasite adherence and cytopathic activity.

Allicin, one of the active principles of freshly crushed garlic homogenates, has a variety of antimicrobial activities. Allicin in its pure form was found to exhibit i) antibacterial activity against a wide range of Gram-negative and Gram-positive bacteria, including multidrug-resistant enterotoxicogenic strains of Escherichia coli; ii) antifungal activity, particularly against Candida albicans; iii) antiparasitic activity, including some major human intestinal protozoan parasites such as Entamoeba histolytica and Giardia lamblia; and iv) antiviral activity. The main antimicrobial effect of allicin is due to its chemical reaction with thiol groups of various enzymes, e.g. alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase, which can affect essential metabolism of cysteine proteinase activity involved in the virulence of E. histolytica.

Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters.

Aspirates of liver abscesses were analyzed for Entamoeba histolytica. PCR detected a gene encoding a 30-kDa protein in all samples but detected the ribosomal DNA gene in only 14 (33.3%) samples. Enzyme-linked immunosorbent assay detected antigen in 41 (97.6%) samples. PCR analysis of a strain- specific antigen (SSG) revealed that abscesses were caused by various strains.

Keywords: GALACTOSE-INHIBITABLE LECTIN; CYSTEINE PROTEINASE GENE; PORE-FORMING PEPTIDE; AMEBIC LIVER-ABSCESS; RIBOSOMAL-RNA GENES; ADHERENCE LECTIN; SURFACE-ANTIGEN; MOLECULAR-CLONING; NONPATHOGENIC ZYMODEMES; MONOCLONAL-ANTIBODIES

Background. Garlic (Allium sativum) has been considered to exhibit therapeutic features for many years. The effects of garlic on levels of serum lipids and on atherosclerosis have been investigated extensively. We have previously demonstrated that allicin, an active component of garlic, exerts a beneficial effect on lipid profile in hyperlipidemic rabbits. Objective. To investigate the effects of allicin on formation of fatty streaks (atherosclerosis) and lipid profile in mice. Methods. Allicin was extracted from garlic and kept in a buffer citrate solution at 4°C. Sixty C57BL/6 mice were fed Paigen diet (17% fat, 1.25% cholesterol) for 15 weeks. Thirty randomly selected animals were administered allicin solution (9 mg/kg) and 30 were administered placebo. Blood lipid profile was evaluated five times during the study. At the end of the 15-week period, the animals were killed and the aortic sinus was evaluated for formation of fatty streaks (atherosclerosis). Results. We observed no statistically significant differences between blood lipid profiles of groups. Microscopic evaluation of aortic sinus formation of fatty streaks (atherosclerosis), however, showed that values for mice in the allicin-treated group were significantly lower: areas of formation of fatty streaks (atherosclerosis) were 13 440 ± 3310 and 23 410 ± 3723 μm², respectively, for allicin-treated and control mice (means ± SEM; P = 0.023). Conclusions. These results indicate that allicin reduces formation of fatty streaks (atherosclerosis) in hyperlipidemic mice. These changes do not seem to occur through an alteration in blood lipid profile.

One of the under-represented genes identified by cDNA representational difference analysis (RDA) between avirulent Entamoeba histolytica strain Rahman and virulent strain HM-1:IMSS was the amoebic light (35 kDa) subunit of the Gal/GalNac lectin complex. This lectin complex, which mediates the adhesion of the parasite to the target cell, also contains a heavy (170 kDa) subunit, which has the carbohydrate-binding domain. Stable transfectants of the virulent strain in which the expression of the 35 kDa subunit was inhibited by antisense RNA were not significantly affected in their adhesion activity to mammalian or bacterial cells but were strongly inhibited in their cytopathic activity, cytotoxic activity and in their ability to induce the formation of liver lesions in hamsters. These findings suggest that the 35 kDa subunit may have a specific function in the pathogenic pathway and provides a new insight into the role of this component of the Gal/GalNac lectin complex in amoebic virulence.

Amoebapores have been proposed to be a major pathogenicity factor of the protozoan parasite Entamoeba histolytica, which is responsible for the killing of target cells. These 77-residue peptides are structural and functional analogues of NK-lysin and granulysin of porcine and human cytotoxic lymphocytes. Inhibition of amoebapore gene expression in amoebae was obtained following transfection with a hybrid plasmid construct (pAP-R2) containing the Neo resistance gene and the gene coding for amoebapore A, including its 5' and 3' untranslated region (UTR) sequences, in reverse orientation under a promoter (g34) taken from one of the E. histolytica ribosomal protein (RP-L21) gene copies. Transfectants of virulent E. histolytica strain HM-1:IMSS, in which the expression of amoebapore was inhibited by ~ 60%, were significantly less pathogenic. Cytopathic and cytolytic activities of viable trophozoites against mammalian nucleated cells, as well as lysis of red blood cells, were markedly inhibited. Moreover, trophozoite extracts of pAP-R2 transfectant displayed lower pore-forming activity and were less potent in inhibiting bacterial growth compared with controls. Notably, liver abscess formation in hamsters by the pAP-R2 transfectant was substantially impaired. These results demonstrate for the first time that amoebapore is one of the pathogenicity factors by which trophozoites of E. histolytica exert their remarkable cytolytic and tissue destructive activity.

Allicin (diallylthiosulfinate) is the main biologically active component of freshly crushed garlic cloves. It is produced upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). A simple and rapid spectrophotometric procedure for determination of allicin and alliinase activity, based on the reaction between 2-nitro-5- thiobenzoate (NTB) and allicin, is described. NTB reacts with the activated disulfide bond S(O)-S of allicin, forming the mixed-disulfide allylmercapto- NTB, as characterized by NMR. The method can be used for determination of allicin and total thiosulfinates in garlic preparations and garlic-derived products. The method was applied for determination of pure alliinase activity and for the activity of the enzyme in crude garlic extracts.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+ S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen- radical generating system [H₂O₂-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by ¹H and ¹³C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP⁺- dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD⁺-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2- ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

The lipophosphoglycan-like (LPG-like) molecules of E. histolytica virulent strains are clearly distinct from those of the avirulent E. histolytica and E. dispar strains. Abundant 'LPG' levels are apparently limited to virulent strains, while lipophosphopeptidoglycans ('LPPG's) are common to both virulent and avirulent strains of E. histolytica and E. dispar. It is therefore conceivable that 'LPPG' performs a function that is essential to survival within the host, while the 'LPG' performs a more specific function related to virulence.

Inhibition of most of the expression of the cysteine proteinases of Entamoeba histolytica strain HM-1: IMSS was successfully performed by transcription of ehcp5 antisense RNA using the promoter of ehg34, which encodes a L21 ribosomal protein of E. histolytica. We have generated a stable transfectant in which the overall level of cysteine proteinase activity is strongly reduced (≃ 90%). This transfectant has a normal growth rate in Diamond's TYI-S-33 medium, a cytopathic and haemolytic activity similar to the control HM-1:IMSS pEhAct-Neo transfectant but with a significantly lower phagocytic activity.

Two genes, EhgLE3 and Ehg34, encoding the ribosomal protein L21 (rp-L21) were identified and characterized from Entamoeba histolytica. Their coding regions are highly conserved, but their flanking regions differ significantly. Analogous genes (EdgLE3 and Edg34) were characterized in E. dispar. The two rp-L21 copies are transcribed at similar levels in the two parasites. However, their relative binding to the polyribosomal complex during active translation is different. In E. histolytica, binding of EhgLE3 transcripts to the polyribosomes is significantly higher in comparison with that of Ehg34 transcripts, whereas in E. dispar the binding pattern is inverse. The importance of each of the rp-L21 flanking regions to gene translation was investigated by constructing hybrid plasmids containing the CAT reporter gene flanked by rp-L21 flanking regions. The plasmids were stably transfected into E. histolytica and E. dispar, and CAT mRNA and enzymatic activity levels were determined, All plasmids promoted transcription of CAT. Yet, in E. histolytica, high levels of CAT activity were observed only when gLE3 upstream regions flanked CAT. In contrast, in E. dispar, high levels of CAT activity were observed when g34 upstream regions flanked CAT. The downstream regions showed no significant effect on CAT translation.

The ability of Entamoeba histolytica trophozoites to destroy monolayers of baby hamster kidney cells is inhibited by allicin, one of the active principles of garlic. Cysteine proteinases, an important contributor to amebic virulence, as well as alcohol dehydrogenase, are strongly inhibited by allicin.

Virulent strains of Entomoeba histolytica have been reported to produce a mixture of phosphoglycoconjugates that share some structural features with the lipophosphoglycans (LPGs) of Leishmania. Purification of these glycoconjugates is essential to their precise structural characterization. In this study we have extracted 'LPG-like' molecules from various virulent E. histolytica strains and purified on the basis of charge differences, 2 apparently related glycoconjugates a 'LPG' and a 'lipophosphopeptidoglycan (LPPG)'. In marked contrast to the abundance of these 'LPG' and 'LPPG' molecules in the virulent strains, avirulent E. histolytica and E. dispar strains produce either very low, or no detectable levels of LPG, and either low levels or modified forms of 'LPPG'. Monospecific polyclonal antibodies prepared against that 'LPG' of the virulent strain HM-1:IMSS c16 identified epitopes shared between both the 'LPG' and the 'LPPG' of this and other virulent strains, using Western blot analysis. Flow cytometric analysis of a range of strains using these antibodies identified a surface distribution of these molecules and confirmed a correlation between surface exposure of epitopes bound by these antibodies and parasite virulence.

A comparison of the use of three commercially available enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes to detect and differentiate Entamoeba histolytica from E. dispar was carried out. Only the Techlab kit did not cross-react with E. dispar antigens, but it was 100 times less sensitive than PCR in detection of and differentiation between the two types of Entamoeba.

One of the three cysteine proteinase genes. ACP₁ (or CP 3), has been reported to be missing in non-pathogenic strains of entamoeba histolytica (or Entamoeba dispar as recently labeled). Unexpectedly, a gene fragment very similar in its sequence (95% homology) to ACP₁ of pathogenic strains was obtained by use of the polymerase chain reaction from genomic DNA and cDNA of various cloned non-pathogenic or E. dispar strains rules out the proposed use of its absence for diagnostic purposes.

Keywords: STRAIN

Gangliosides were found to be present in Entamoeba histolytica. They were extracted from lyophilized trophozoites of the pathogenic strain HM-1:IMSS and purified by high performance thin-layer chromatography. Two resorcinol-positive bands, comigrating with GM2 and GD1a were demonstrated, revealing the existence of ganglioside molecules in Entamoeba histolytica. The GM2 content, determined as lipid-bound sialic acid, was 1.5 μg/10⁸ amoebae, the content of the GD1a comigrating band was 0.32 μg/10⁸ amoebae. The identity of the GM2 comigrating band was confirmed by TLC immunostaining, using the monoclonal anti-GM2 antibody GMB28. Furthermore, six out of ten anti-amoeba positive sera selectively reacted with the GM2 comigrating band, as revealed by immunostaining on TLC plates. Absorption tests revealed that preincubation of anti-amoeba positive sera with standard GM2 was followed by a significant decrease in the reaction with amoeba trophozoites by indirect immunofluorescence. These results demonstrate that a GM2 comigrating component of Entamoeba histolytica may be one of the antigens responsible for the appearance of circulating antibodies in patients with amoebiasis.

Alliinase (EC 4.4.1.4) catalyses the production of allicin (thio-2-propene-1-sulfinic acid S-allyl ester), a biologically active compound which is also responsible for the characteristic smell of garlic. It was demonstrated that alliinase which contains 5.5-6% of neutral sugars, gives clear PAS-staining, binds to Con A and can form a complex with garlic mannose-specific lectin (ASA). Evidence that the formation of such a complex is mediated by the interaction of the carbohydrate of the glycoprotein enzyme with the lectin was obtained from a radioligand assay which demonstrated the binding of alliinase to ASA and competitive inhibition of this binding by methyl α-d-mannoside. ASA I was shown as the lectin mainly present in the complex with alliinase. The results of this study also demonstrate that alliinase is glycosylated at Asn¹⁴⁶ in the sequence Asn¹⁴⁶-Met¹⁴⁷-Thr¹⁴⁸.

Background: The effect of garlic on the serum lipid profile has been the subject of controversy. This study was therefore designed to examine the effects of allicin, an active constituent of garlic, on the lipid profile in a rabbit model. Methods: Allicin was produced by reacting alliin, synthesized in our laboratory, with purified alliinase. Nineteen New Zealand White rabbits fed a cholesterol-rich diet (0.25% cholesterol) for 18 weeks. Ten rabbits received freshly produced allicin (3 mg/kg orally) starting at 8 weeks, and nine received placebo. There was no significant difference between the lipid profiles of the two groups at baseline up to 8 weeks. Results: From day 28 of allicin supplementation a significant difference was found between the allicin and placebo groups in the graph regression lines describing the influence of allicin on serum cholesterol: Y = 41.39 + 8.69 multiplied by day (control) versus Y = -877.24 + 17.67 multiplied by day (allicin). The same trend was found for low-density lipoprotein concentrations: Y = 10.3 + 8.4 multiplied by day (control) versus Y = -750.4 + 15.7 multiplied by day (allicin). The serum high-density lipoprotein levels also differed significantly between the groups: Y = 20.29 + 0.24 multiplied by day (control) versus Y = -109.9 + 1.65 multiplied by day (allicin). Conclusions: Our results indicate that allicin has a beneficial effect on the serum lipid profile in hyperlipidemic rabbits, and should be further tested clinically.

A monoclonal antibody (MAb), 318-28, that specifically reacts with a 30- kDa antigen present on membrane surfaces of all nonpathogenic (NP) Entamoeba histolytica strains tested and which did not react with pathogenic (P) strains was used for the isolation of the cDNA coding for this antigen from an expression library of an NP E. histolytica strain. The deduced amino acid composition was rich in lysine residues (14.5%), with some sequence similarity to a polyadenylate-binding protein. Southern and Northern (RNA) blot analyses, as well as amplifications of DNA segments by PCR, indicate that a very similar gene (identity of 96.5%) exists in P strains of E. histolytica. Unexpectedly, the NP-specific antigen was also identified by MAb 318-28 on the surfaces of a cloned, xenically cultivated and well- characterized P strain (BNI:0591) that was recently isolated from a human liver abscess. Binding of the MAb, both to the cell surfaces and to Western blots (immunoblots), was abolished, however, upon axenization of the BNI:0591 cultures. Oligonucleotide primers, designed to anneal only to specific DNA sequences of the NP 30-kDa protein gene copy, amplified a DNA segment from P strain BNI:0591 which was identical in sequence to that of the NP 30-kDa protein gene. Our findings indicate that a P strain of E. histolytica can possess and express, under certain growth conditions, an antigen that is usually detected only in NP strains.

It is known that human serum contains natural antibodies to self and non-self proteins. We wished to determine whether normal human serum contains antibodies to dietary proteins that were never injected. We found that human serum contains antibodies to the two major proteins from cloves of garlic (Allium sativum) which is used as a flavorigard dietary food additive. The antibodies found were directed against alliinase and mannose-specific Allium sativum agglutinin (ASA). The antibodies were purified by affinity chromatography on their corresponding antigens. The purified immunoglobulins were mainly of the IgG and IgM classes and could be divided into two categories - specific and crossreactive. The anti-alliinase antibodies were highly specific, while anti-ASA antibodies were polyreactive. Some of the possible reasons for this difference in specificity are suggested.

A second gene (rp-L21) copy, clone g34, coding for ribosomal (r-) protein L21, was isolated from the pathogenic (P) strain HM-1:IMSS c16 of the intestinal parasite Entamoeba histolytica (Eh). The gene was compared to the previously isolated copy, gLE3 [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], with respect to its primary structure, mRNA levels and binding to the r-complex during translation. Unlike the gLE3 gene copy [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], g34 was found not to be physically connected to an actin gene copy. Homologous copies of the two rp-L21 genes were also characterized from the nonpathogenic (NP) strain SAW1734R clAR, as well as from its P derivative. Sequence comparison of the coding regions of the two rp-L21 revealed almost full identity. Significant differences were found, however, within their 3' and 5' flanking regions. Using the 3' rapid amplification of cDNA ends (3' RACE) method [Frohman et al., Proc. Natl. Acad. Sci. USA 85 (1988) 8998-9002], as well as Northern and slot blot hybridizations, it was demonstrated that both rp-L21 mRNAs are found in similar amounts. However, as was shown by differential hybridization, the relative binding of each transcript to the r-complex varied somewhat between P and NP strains. This finding suggests that the control of expression of rp-L21 in Eh may involve regulation at the post-transcriptional level.

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its K _m using synthetic S-allylcysteine sulfoxide (+isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N- terminal of the intact polypeptide. The second step involved screening of garlic λgt11 and λZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3 noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from a wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.

Studies on the interaction between trophozoites of Entamoeba histolytica of pathogenic or nonpathogenic origin and epithelial cells of the human intestine can contribute to the understanding of the pathogenesis of invasive amoebiasis. We have examined the interaction of virulent E. histolytica with the human colonic carcinoma cell line HT-29. Differentiated HT-29 cells are comparable to the mucosa cells to which E. histolytica attaches physiologically. Adherence between E. histolytica trophozoites and HT-29 cells was effectively inhibited by glycoconjugates containing galactose, indicating the importance of the 170-kDa lectin of E. histolytica in binding to intestinal cells. Adherence was not significantly inhibited by glycoconjugates containing N-acetyl-glucosamine, indicating that the 220-kDa lectin of E. histolytica is not involved in binding to HT-29 cells. The destruction of HT-29 cells by pathogenic E. histolytica was dependent on adherence. The destruction was enhanced when polymorphonuclear granulocytes were added to the E. histolytica trophozoites.

The electrophoretic karyotypes of a pathogenic and a nonpathogenic strain of Entamoeba histolytica were determined by pulsed-field gel electrophoresis. A number of previously isolated genes were assigned to specific chromosomal bands. Significant differences between the chromosomal patterns of these strains as well as in the assignment of most genes were found.

Shigella flexneri M90T (invasive) and BS176 (noninvasive) are typical nonfimbriated organisms that do not bind to or activate phagocytic cells. We demonstrate that S. flexneri M90Tp and BS176p, obtained by transformation of the strains named above with the cluster of genes encoding type 1 (mannose- specific) fimbriae of Escherichia coli, express the functional fimbriae, as shown by electron microscopy, by binding of antifimbria antibodies and by yeast cell aggregation. The transformants, but not the parental strains, bound to human granulocytes and mouse peritoneal macrophages. This binding was inhibited by methyl α-D-mannoside but not by methyl α-D-galactoside. The bound bacteria induced oxidative burst activation and degranulation of the granulocytes in vitro. With mouse peritoneal macrophages, the binding of the fimbriated bacteria induced degranulation in vitro. Injection of the bacteria into mouse peritoneum also induced degranulation of the macrophages in vivo; no such effect was observed with the nonfimbriated strains. The bound fimbriated transformants were effectively killed by the human granulocytes in vitro in the absence of opsonins or after opsonization with human anti-S. flexneri antiserum. The nonfimbriated strains were killed only after opsonization. These results provide further evidence for the role of type 1 fimbriae in lectin-mediated nonopsonic phagocytosis.

The establishment of a relationship between Entamoeba histolytica and certain bacteria may contribute to the expression and/or enhancement of the pathogenicity of this parasite. Recent experiments have shown that bacteria expressing mannose-binding lectins on their surface could attach to mannose-containing molecules on the surface of amoebae. In this study, we established a model of interaction between. E. histolytica and Shigella flexneri. Using well-characterized mutants of S. flexneri, we studied the role of type I pili expression and the invasive phenotype of S. flexneri in the interaction between amoebae and the bacteria. Type I pill expression allowed attachment and subsequent internalization of S. flexneri by amoebae, these events were not observed in isogenic strains that did not express type I pili. Invasive as well as non-invasive variants of S. flexneri expressing type I pilin were slowly digested by amoebae following internalization. Morphological studies showed that the specific features of the interaction depend on the dynamics of the distribution of mannose residues on the amoebic membrane during the interaction.

Twenty Entamoeba histolytica strains from patients with HIV-1 infection were isolated and compared with E. histolytica strains from patients without HIV infection. The isoenzyme pattern of the hexokinase as well as the hybridization with known DNA probes were used as markers for pathogenicity. According to these markers, all 20 strains could be regarded as being nonpathogenic. Direct measurements of the virulence were carried out: destruction of monolayer tissue culture cells, capacity of phagocytosis and the ability to induce liver abscesses in the hamster. The virulence of strains from HIV patients was comparable to that of E. histolytica strains which had been isolated from HIV-negative asymptomatic carriers. In agreement with this, none of the HIV-positive patients showed symptoms of an invasive amebiasis. By PRC, no HIV-1 proviral DNA could be evidenced in the E. histolytica strains which had been isolated from HIV patients.

The coding sequence deduced from two overlapping cDNA inserts obtained from a pathogenic strain of Entamoeba histolytica revealed a striking homology (>85%) with elongation factor EF-1α from Saccharomyces cerevisiae and Artemia salina. The deduced amino acid sequence predicted a size of 49 kDa, and antibodies raised against the S. cerevisiae EF-1α cross-reacted with an amoebic protein of similar size (45-47 kDa). Sequence analysis of the cDNA revealed that the 5 untranslated region contained a stretch of 190 nucleotides which was perfectly complementary to a segment of the 3 terminal coding region situated 1015 bases downstream of the methionine initiation codon. Electron microscopy of self-renatured cDNA confirmed the potential of such molecules to form a stem-loop secondary structure. The presence of the complementary sequences was confirmed at the genomic level by sequence analysis of polymerase chain reaction-amplified segments which span both the 3 and 5 terminal complementary regions. Comparison of the deduced amino acid sequence of E. histolytica EF-1α with Ef-Tu from Escherichia coli and EF-1α from different sources, suggested that the major functional domains of the protein are located within the loop structure.

Entamoeba histolytica possesses a 24.5 kilobase plasmid-like molecule which encodes for the organism's ribosomal RNAs. Sequence analysis of this extrachromosomal element revealed the presence of AT rich sequences which show homology to the origin of replication of other lower eucaryotes. An 802 bp fragment containining these sequences was cloned into a yeast shuttle vector lacking the origin of replication and the construct tested for its ability to replicate autonomously in yeast. Mitotic stability tests as well as evidence for plasmid maintenance indicate that the transformed cells contained self-replicating episomes and not stably integrated molecules. The nucleotide sequence of this ARS-containing fragment is presented.

Keywords: Biochemistry & Molecular Biology; Genetics & Heredity; Immunology; Parasitology; Pharmacology & Pharmacy

Growth of Entamoeba histolytica trophozoites was inhibited by 50% at low concentrations (2.0 μg/ml) of the diazopeptidyl inhibitor benzyloxycarbonyl-leucyl-L-tyrosyldiazomethane (Z-L-Leu-L-Tyr-CHN₂). Iodination of the tyrosine residue lowered the growth inhibitory efficacy of the diazopeptidyl inhibitor (50% inhibition, approximately 10 μg/ml). However, even at this concentration, practically all of the cysteine proteinase activity of the cells was irreversibly inactivated as shown by fluorescence microscopy with the dipeptide substrate L-Arg-L-Arg-4-methoxy-β-napthylamide or colorimetrically with azocasein as the substrate. Growth of trophozoites of E. histolytica from various strains, including both pathogenic and nonpathogenic zymodemes, was similarly inhibited. The concentration of inhibitor required to inactivate the proteinase activity of nonpathogenic cells was lower. Lysates from trophozoites grown in the presence of sublethal concentrations of ¹²⁵I-labeled protease inhibitor (10 μg/ml) showed as many as eight radioactive bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular sizes, 73, 68, 56, 40, 39, 35, 29, and 27 kilodaltons), Two of these bands (molecular sizes, 29 and 27 kilodaltons) could be seen in gels of the cytoplasmic fraction, whereas the high-molecular-size bands were mostly associated with the membrane fraction. The radioactive bands in pathogenic and nonpathogenic strains were very similar with only minor differences. The results obtained show that E. histolytica cells, irrespective of their pathogenicity, possess a number of cysteine proteinases of similar molecular sizes which are vital for cell growth.

1. 1. Protein kinase C (PKC) activity has been identified in various strains of the human parasite, Entamoeba histolytica. 2. 2. An amoebic protein of mol. wt 78,000 was recognized by polyclonal antibodies raised against the 82,000 mol. wt rat brain protein kinase C. 3. 3. A partially purified PKC preparation from E. histolytica phosphorylated histone I in the presence of calcium, phopholipds and diacyglycerol, and specifically bound tritiated phorbol ester at an apparent K_D of 9 nM. 4. 4. A relocalization of the amoebic PKC activity from the cytosol to the membrane fraction was observed when trophozoites were actively phagocytising bacteria. Under these conditions, a labelled phospho-protein of mol. wt 68,000 was identified. 5. 5. Similar to what was found during macrophage activation, a myristoylated mol. wt 68,000 protein was detected in amoebae grown in the absence of bacteria, but not in amoebae which were active in phagocytosis.

Most individuals infected with Entamoeba histolytica are reported to be clinically asymptomatic. On the basis of the electrophoretic migration of hexokinase and phosphoglucomutase isoenzymes, two groups of E. histolytica isolates have been classified. Those derived from symptomatic cases were found to have fastmigrating hexokinase bands and were labeled pathogenic. The others, isolated from cyst passers, had (in most cases) slow-migrating bands and were called nonpathogenic. Differences between these two groups of E. histolytica were found recently at the DNA level. Two sets of different DNA probes derived from tandemly repeated sequences present in extrachromosomal circular DNA elements in each group of E. histolytica were characterized. Using these probes with procedures for direct hybridization of trophozoites on nylon membranes, we could correctly correlate hexokinase electromobility with the DNA hybridization signal of 81 different isolates of E. histolytica. The advantages of using DNA probes lie in their sensitivity (fewer than 200 throphozoites can be detected) and specificity. The probes hybridized only with amebae from the E. histolytica species and not with other enteric protozoa and can thus be useful as a diagnostic tool.

Strains of Entamoeba histolytica which were isolated from symptomatic patients and which possess a characteristic pathogenic isoenzyme pattern (zymodeme) have extrachromosomal circular DNA molecules containing RNA genes and clusters of tandemly reiterated PvuI elements. The nucleotide sequence of comparable reiterated BamHI elements present in amebae with nonpathogenic zymodemes differs from that found in pathogenic ones. By using the polymerase chain reaction, it was demonstrated that the cloned, nonpathogenic E. histolytica strain SAW 1734R clAR also contains one or few of the tandemly repeated DNA PvuI elements characteristic of the pathogenic amebae. Sequences were detected by hybridization with the P-145 probe after in vitro amplification. Because of technical difficulties, it was impossible to resolve whether single copies of the nonpathogenic BamHI repetitive elements are present in pathogenic amebae. Our findings suggest that in the nonpathogenic amebae, the signal to start amplifying the PvuI-type elements may be induced during the process of elimination of bacterial associates from their growth environment.

Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patients with invasive disease have characteristic isoenzyme profiles (pathogenic zymodemes), suggesting a role for parasite factors in determining the severity of infection. A galactose-specific cell surface lectin from a pathogenic zymodeme was shown to mediate in vitro adherence to human colonic mucins and contact-dependent killing of target cells. Six nonoverlapping antigenic determinants were identified on the 170-kilodalton heavy subunit of the pathogenic lectin. Anti-lectin monoclonal antibodies (MAb) directed against epitopes 1 and 2 enhanced adherence whereas MAb to epitopes 3 through 6 either inhibited or had no effect on adherence. We tested 50 pathogenic and nonpathogenic strains for reactivity to these anti-lectin MAb by radioimmunoassay. MAb to epitopes 1 through 6 reacted in the radioimmunoassay with all 16 pathogenic zymodeme strains tested. In contrast, only MAb to epitopes 1 and 2 bound to the lectin from nonpathogenic strains. Western immunoblots with anti-lectin antibodies showed that the 170-kilodalton heavy subunit was present in the nonpathogenic amebae. Adherence of the nonpathogenic SAW 760 strain to human erythrocytes was enhanced by MAb to epitope 1 and blocked by galactose, confirming the presence of a functionally active lectin. A lectin radioimmunoassay based on MAb to epitopes 1 and 3 proved to be a simple and rapid method to distinguish pathogenic from nonpathogenic amebae in culture. Further exploration of the functional consequences of the antigenic differences demonstrated for the lectin may lead to a better understanding of its role in pathogenesis.

Most commonly used antiamoebic drugs are effective in invasive amebiasis, but their response against trophozoites of Entamoeba histolytica, present in the lumen of the human colon, is inadequate. We report the development of an antiamoebic drug carrier that may be effective against luminal infections. Our preparation consists of small silica particles (5-10 Ilm in diameter) covalently linked to a potent antiamoebic drug, 2-(4-aminophenoxymethyl)- 5-nitro-I-methyl imidazole. Silica-drug particles were injected into mice, hamsters, and guinea pigs. We found that trophozoites phagocytosed the particles in vivo and in vitro, followed by rapid cell death due to the released drug. Analysis of mouse serum revealed that no drug was absorbed from the intestine after placement of the drug-containing particles in the intestine. The antiamoebic activity of particles recovered from the intestine was almost fully retained. This novel antiamoebic concept may be useful for luminal therapy for asymptomatic amebiasis and may minimize side effects and frequency of administration.

Highly abundant DNA fragments obtained after restriction enzyme digests of nuclear DNA of Entamoeba histolytica strain HM-1:IMSS have been cloned and characterized. Northern blot hybridization to E. histolytica rRNA and sequence analysis identified the abundant DNAs as ribosomal DNA containing species. Several overlapping clones containing these abundant DNAs were isolated from 4 different genomic libraries of E. histolytica. Alignment of the restriction maps was consistent with a circular molecule, about 24.6 kilobase pairs (kb) in size. Nuclease BA131 digestion provided additional evidence for the circular nature of this DNA. The ribosomal DNA molecule contains two large inverted repeat-regions, each at least 5.2 kb in length. Sequence analysis of clone R715 revealed homology to the large rRNA units of various eukaryotic organisms. This clone was located in both inverted repeats, suggesting two rRNA cistrons per molecule. The inverted repeats are flanked by stretches of DNA which contain tandemly reiterated sequences. Southern blot analysis of E. histolytica nuclear DNA revealed the presence of two populations of molecules. These molecules have identical arrangements of restriction sites, but differ in size (0.7 kb) in a fragment containing tandemly reiterated sequences. Analysis of E. histolytica nuclear DNA by electron microscopy also revealed circular molecules. These molecules are about 26.6 kb ± 0.5 kb in size and contain structural features predicted by the restriction map of the extra-chromosomal ribosomal DNA of E. histolytica.

A number of DNA probes which hybridize to highly abundant DNA sequences of Entamoeba histolytica were developed. Variations in the hybridization patterns of different E. histolytica strains were detected with selected probes. Four types of restriction fragment length patterns were obtained. Of these, the first class belonged to E. invadens and E. histolytica-like var. Laredo. The next two classes consisted of various strains of E. histolytica which were originally isolated from symptomatic patients and possessed pathogenic patterns of isoenzymes (zymodemes), whereas the fourth group contained E. histolytica strains with nonpathogenic zymodemes obtained from asymptomatic carriers. DNA probes, based on DNA sequences specific to E. histolytica isolates with pathogenic and nonpathogenic zymodemes were isolated, and their nucleotide sequences were determined. These probes (P145 and B133) hybridized selectively to DNA of isolates possessing either pathogenic or nonpathogenic isoenzyme patterns. The newly developed probes could be useful for diagnostic purposes and could serve as tools to investigate the molecular basis of pathogenicity and the genetic mechanisms which regulate the variable aggressive behavior of the parasite.

A cDNA clone (subclone B) previously isolated from the human parasite Entamoeba histolytica was characterized. DNA sequence analysis of subclone B identified the DNA as that encoding apoferredoxin. E. histolytica ferredoxin cDNA contains unusually short 5 and 3 noncoding regions of 9 and 25 nucleotides, respectively. A genomic ferredoxin clone was isolated from E. histolytica DNA, and comparison of genomic and cDNA sequences revealed that the ferrodoxin gene is unspliced. The deduced amino acid sequence of E. histolytica ferredoxin resembles clostridial type of ferredoxins, and shows an arrangement of cysteines characteristic for the coordination of 2[4Fe-4S] centres. Of interest is the absence of an aromatic amino acid in the N-terminal region of the protein, a feature which is conserved in clostridial ferredoxins. Southern blot analysis of three different E. histolytica strains (200:NIH, Rahman and HM-1:IMSS) demonstrated the presence of a family of at least two ferredoxin genes. One of these genes is marked by restriction length polymorphisms in different strains of E. histolytica.

The pathogenic potential of four Entamoeba histolytica isolates obtained from asymptomatic carriers and possessing nonpathogenic zymodemes was compared to four E. histolytica strains obtained from invasive cases of amebiasis and having pathogenic zymodemes. Both xenic and axenic cultures of a number of strains were tested. Determinations of cytopathogenicity were done in vitro by measuring the rates of destruction of tissue cultured monolayers of baby hamster kidney cells by intact amebae or by its cell-free extracts. The in vivo virulence was tested by assessing their capacity to form hepatic abscesses in hamsters or cecal ulcerations in rats. The results obtained show that two of the isolates from asymptomatic carriers (strains SAW 1734R clAR and WI:0385:191) were as virulent as three of the invasive ones (HM-1:IMSS, 200:NIH, and SAW 408). Two other isolates from asymptomatic carriers and one from a dysentery case were avirulent. All the E. histolytica isolates tested were similarly sensitive to metronidazole and emetine (IC₅₀ 1-10 μg/ml). The results indicate that the pathogenic potential of E. histolytica varies between isolates and can be affected by culture conditions and by the presence or absence of bacterial cells. These findings suggest that virulence does not necessarily correlate with a pathogenic zymodeme.

Amoeba-bacterium cultures of Entamoeba histolytica transferred to a hypoosmotic medium depleted of nutrients changed morphologically and biochemically. The cells ejected strains of rice starch, rounded up, and formed a distinct cell wall that was resistant to detergent, bound the sialic acid-specific lectin from Limulus polyphemus, and became fluorescent with Calcofluor M2R. A subpopulation of these cells displayed more than one nucleus. All these signs are characteristic of encysting cells and were also observed in cysts obtained from a human patient. The morphological changes were accompanied by the appearance of two new glycoproteins with apparent molecular sizes of 100 and 150 kilodaltons which contained sialic acid. Sialic acid has been reported to be absent from trophozoites of Entamoeba species. The presence of this sugar residue on cyst-specific proteins parallels recently reported findings during the encystation of the related reptilian parasite Entamoeba invadens. This may indicate a basic role for sialic acid in the encystation of Entamoeba parasites.

A novel sialoglycoprotein with an apparent molecular mass of approximately 250 kDa was detected on the surface of cysts of Entamoeba invadens. Sialic acid was identified in this glycoprotein by gas chromatography after methanolysis; N-acetyl- and N-glycolyl neuraminic acid were identified by thin layer chromatography in hydrolysates of partially purified preparations of the 250 kDa glycoprotein as well as in whole cysts. The sialoglycoprotein is stage-specific and could be detected by binding of wheat germ agglutinin and a specific monoclonal antibody (JAM3) only to precysts and mature cysts but not to trophozoites. A 250 kDa protein could be metabolically labeled with [³⁵S]methionine. This, together with the absence of such a glycoprotein in the encystation medium, suggests that the 250 kDa sialoglycoprotein is not an adsorbed serum glycoprotein. Indirect evidence suggests that the parasite may utilize serum components as a source for sialic acid.

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8-5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48 000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.

The protecting effect of human milk against intestinal infections has been well documented, but its mechanism not completely understood. We have examined the effect of the nonimmunoglobulin fraction (NIgF) of human milk and colostrum on bacterial adherence to the intestinal tract. The NIgF was prepared by passing the milk through an immunosorbent column containing rabbit antihuman γ-globulin (IgG and IgA). The effluent fraction did not contain γ-globulins as shown by immunodiffusion on agarose and by using rabbit antihuman Ig, that was then detected with fluorescently-labeled goat antirabbit Ig. The effect of the NIgF of human milk on the adherence of enterotoxigenic Escherichia coli strains to guinea pig intestinal tract was quantitatively determined using radiolabeled bacteria which were incubated with suspensions of viable intestinal cells. Thirteen to 17 bacteria adhered per intestinal cell. NIgF of human milk and colostrum (300 fil, 6.7 mg) caused about 50% inhibition of the adherence of enterotoxigenic E. coli strains whose attachment was mediated by colonization factor antigen I and II. No inhibition was noted on the adherence of enterotoxigenic E. coli strains containing type I pili. The inhibitory activity resisted boiling and proteolytic digestion with trypsin, but was completely abolished by periodate treatment, indicating that carbohydrate residues were probably involved. Examination of the effect of NIgF of human milk on bacterial adherence to intact intestinal surfaces revealed comparable results. Observations with scanning electron microscopy confirmed, morphologically, the attachment of the bacteria and the inhibitory effect of human milk. It is concluded that receptor-like glycocompounds in human milk and colostrum inhibit the adherence of certain enterotoxigenic E. coli strains to the intestinal mucosa. This may play a role in protecting infants against intestinal infections.

In order to study gene expression in the human parasite Entamoeba histolytica, a cDNA library of E. histolytica strain 200: NIH was constructed using the phage vector λgt10. Three cDNA clones (A,B and C) were selected for further analysis. Each of the three clones hybridized to a distinct mRNA. Two of these mRNAs were translated in vitro afer hybrid selection, and yielded distinct translation products. One of these mRNAs, selected by hybridization to clone A, encodes the most abundantly expressed protein in E. histolytica. DNA sequence analysis of this cDNA clone identified the DNA as that encoding actin. The deduced amino acid sequence of E. histolytica actin resembles both cytoplasmic and muscle actins and has unusual N-terminal glycine residue. We have shown that a family of actin genes is present in E. histolytica actin clones were obtained from a λgt10 genomic library using subcloned cDNA probes. Southern analysis of three different E. histolytica strains (200:NIH, Rhaman, and HM-1:IMSS) revealed at least four different actin genes. Strain HM-1:IMSS, however, differs by the presence of an additional actin gene.

Adherence of bacteria via their surface lectins to host epithelial cells is considered an important initial event in bacterial pathogenesis. Mannose-specific (type 1) fimbriae are among the most commonly found lectins in enterobacteria. We studied the effect of aromatic α-glucosides of mannose on the agglutination of mannan-containing yeasts by different strains of Escherichia coli and on the adherence of the bacteria to guinea pig ileal epithelial cells. In both systems these compounds were considerably more effective inhibitors than methyl α-mannoside, with 4-methylumbelliferyl α-mannoside and p-nitro-o-chlorophenyl α-mannoside being the strongest inhibitors. Both compounds were approximately 400-times stronger inhibitors of yeast agglutination by E. coli O128 than was methyl α-mannoside and 1,000- and 470-fold stronger, respectively, than was methyl α-mannoside in inhibiting the adherence of the bacteria to ileal epithelial cells. 4-Methylumbelliferyl α-mannoside was 540 to 1,000 times more effective in inhibiting yeast agglutination by four additional strains of mannose-specific E. coli. It was also more efficient than methyl α-mannoside in removing adherent E. coli O128 from ileal epithelial cells. Our resuls provide further evidence that type 1 fimbriae of E. coli possess a hydrophobic region next to the mannose-binding site. The results suggest that 4-methylumbelliferyl α-mannoside and p-nitro-o-chlorophenyl α-mannoside are good candidates for the design of therapeutic agents that may prevent adherence in vivo and infection by E. coli strains that express type 1 fimbriae.

It is generally recognized that there are nonpathogenic and pathogenic strains of Entamoeba histolytica and that the differences between them may be related in part to the type of bacterial species with which they become associated in the human intestine. The development of methods for cultivating the parasite axenically in vitro have made it possible to study the interactions between amebae-bacteria and mammalian host cells. Our present knowledge of the mechanisms and molecules involved in the intercellular recognition and the effect they have on virulence of E. histolytica, as well as on the relation between isoenzyme patterns (zymodemes) and pathogenicity, is critically reviewed and discussed.

ABSTRACT. Trophozoites of Entamoeba invadens IP1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60160 mosmol/kg. Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg. Encystation could be obtained in the absence of serum although higher yields were obtained in its presence. No difference in the yield of mature cysts was found when either dialyzed or full serum was used. High yields of encystation were obtained (>70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO₄, suggesting that the mechanism of encystation is not induced via sodium or potassium channels. Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation. A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.0611.073 g/ml) from growth medium to the low osmotic pressure encystation solutions. Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml. As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.0491.061 g/ml.

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

The axenization of an Entamoeba histolytica isolate with a nonpathogenic isoenzyme electrophoretic pattern (zymodeme) was recently achieved for the first time (15). Forty days after the cells were transferred to the medium used for axenic cultivation, the amebae developed virulence properties, and the zymodeme converted to a pathogenic pattern. To exclude the possibility that the original isolate consisted of two zymodeme populations and that conditions of growth selected for a particular population, the experiment was repeated with a cloned culture of a nonpathogenic (zymodeme III) strain, E. histolytica SAW 1734R clAR, isolated by and obtained from P.G. Sargeaunt. Axenization was accomplished, as before, by transferring trophozoites to TYI-S-33 medium containing a mixture of antibiotics to suppress the growth of the associated bacterial flora and a nutritional supplement consisting of γ-irradiated bacteria. A change in the hexokinase and phosphoglucomutase isoenzyme pattern was observed 21 days after the amebae had been transferred to the axenic medium but before complete axenization of the amebae had occurred. The change in zymodeme was accompanied by an increase in virulence, as evidenced by the ability of fewer amebae to induce hepatic abscesses in hamsters. A reverse conversion to a nonpathogenic zymodeme was also accomplished by reassociating and subculturing the newly converted pathogenic trophozoites of strain SAW 1734R clAR with the bacterial flora that accompanied this ameba in the original xenic culture. The electromobilities of the hexokinase isoenzymes changed back to their original pattern 7 days after the amebae were returned to xenic growth conditions. Our in vitro results demonstrate that culture conditions and bacterial flora can cause changes in the zymodeme and virulence of a cloned ameba isolate and raise the concern that this could happen also in vivo. Thus, the finding of a particular zymodeme in a culture of E. histolytica isolated from a carrier should not be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Changes in cell surface components of axenically grown trophozoites of Entamoeba invadens which occur during encystation were followed. Protein patterns of trophozoites, immature and mature cyst forms, were analyzed by sodium dodecyl sulphate gel electrophoresis. Total protein profiles of trophozoites and cyst forms stained by Coomassie blue gave similar patterns. In contrast, a number of different bands were observed in gels stained with the carbohydrate-specific Schiff's reagent as well as when nitrocellulose blottings were treated with ¹²⁵I-radiolabelled wheat germ or soybean agglutinins. The most notable differences were bands at 250 and 95-105 kDa present in the cyst forms and absent in the trophozoites, and two bands at 70 and 75 kDa present in the latter and missing in the cysts. Labelling of trophozoites and cyst cell surfaces by iodination with lactoperoxidase revealed a number of protein bands which were exposed on the trophozoite surface and missing in the cysts. Moreover, gel electrophoresis patterns of non-reduced or reduced samples also differed considerably, indicating that a number of proteins are linked by disulphide bonds. This study shows that specific glycoproteins are produced during cyst formation.

Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200x) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain Escherichia coli strains.

The antibacterial activity of canine pancreatic fluid was investigated in an attempt to understand the resistance of this organ, when intact, to ascending bacterial infections. The pancreatic fluid demonstrated bactericidal activity against Escherichia coli, Shigella species, Salmonella species, and Klebsiella pneumoniae; bacteriostatic activity against coagulase-positive and coagulase-negative staphylococci and Pseudomonas aeruginosa; and fungistatic activity against Candida albicans. There was no demonstrable antibacterial activity against Bacteroides fragilis and Streptococcus faecalis. The antibacterial activity was dialyzable and pH dependent, but independent of heat, the activity of several digestive pancreatic enzymes, and the bacterial inoculum. Electron micrographs of Escherichia coli exposed to pancreatic fluid did not demonstrate changes in the bacterial cell wall. Tracer studies of susceptible bacteria demonstrated decreased leucine uptake when briefly exposed to pancreatic fluid. The antibacterial activity was found by column chromatography to be a small molecular peptide. It is likely that pancreatic antibacterial factors protect the pancreas from ascending bacterial infections and operate along with other factors in the homeostasis of the upper small bowel flora.

The adherence of bacteria to pediatric IV catheters and needles was studied. Scanning electron micrographs showed that bacteria adhered well to the catheters and needles, mainly to non-smooth surface areas. In vitro quantitative determination, with the use of radiolabeled bacteria, revealed differences in the affinity of bacteria for the various IV cannula materials. The adherence per square area was greatest for plastic catheters, less for steel needles, and least for siliconized needles. Mean values for the adherence of Staphylococcus aureus to these cannulae were 37.9-40.3 x 10⁵ bacteria/cm² for the plastic catheters; 10.2 x 10⁵ bacteria/cm² for the steel needles, and 7.2-7.6 x 10⁵/cm² for the siliconized needles. Removal of the glutaraldehyde-fixed bacteria adhered to the cannulae, after their placement in veins of rabbits, was lower for the plastic catheters than the IV needles. The appearance and severity of venous phlebitis produced by the various cannulae was determined in an animal model. The degree of the inflammatory response elicited correlated with the in vitro bacterial adherence, indicating that bacterial adherence plays a role in the appearance of cannulaassociated phlebitis. In view of our results and other previous observations of lower rate of infections with the use of IV needles, it is suggested that needles should be preferred to plastic catheters whenever possible. The described in vitro assay for bacterial adherence can be used to determine the adherent properties of IV cannulae, which should be considered in any future cannula design.

Since postnatal development of the gastrointestinal tract has an important effect on its microbial flora and may influence the types of intestinal infections, we examined the effect of age on bacterial adherence to intestinal epithelial cells. Radiolabeled bacteria were incubated with guinea pig enterocytes released by treating loops of the intestine with solutions containing EDTA, dithiothreitol, and citrate. Nonbound bacteria were separated from intestinal cells by sedimentation on a Percoll gradient. The colonic cells avidly bound Shigella flexneri(64 bacteria per cell), Escherichia coli0124 (59), and E. coli 0128 (53). The adherence process was Ca²⁺and temperature dependent, was inhibited by fucose, glucose, and mannose, and was shown to be mediated by a carbohydrate-binding protein (lectin) on the colonic cells. Adherence of these bacteria to intestinal cells of newborn animals was only 15- 25% of the adherence to adult animal cells and increased gradually, reaching adult values at about 2 weeks of age. The lectin activity, which was determined by agglutination of bacteria, was secreted with the colonic mucus. It was undetectable in the newborn animal, appeared gradually with age, and its titer correlated with the adherent capability of the colonic cells.E. coli0128 was the only one of the bacteria tested which significantly adhered to the ileum (19 bacteria per cell) in a process inhibited by mannose. This adherence was mediated by a mannose-sensitive lectin in the bacterial pili, and not on the intestinal cells. The postnatal age had no effect on the adherence to the ileum; the newborn animal had the same adherence capability as the adult one. Good correlation was found between the adherence to the suspended intestinal cells and the adherence to intact intestinal surfaces or the in vivoadherence to intestinal loops.These patterns of bacterial adherence can be important in the neonatal microbial colonization of the intestinal tract, and can play a role in the types of intestinal infections during the neonatal period and infancy.

Treatment of several tumor cell lines, including the murine melanomas B16 and S91 and the human sarcoma Hs791 and Hs705, with retinoic acid resulted in an increased sialylation of specific cell surface membrane sialoglycoproteins. This treatment also augmented the sensitivity of these cells to the cytopathic effects of a sialic acid-specific toxin from Entamoeba histolytica. In contrast, a similar treatment with retinoic acid of a retinoic acid-resistant mutant clone S91-C154, which does not increase sialylation of cell surface glycoproteins, failed to alter the susceptibility of the cells to the E. histolytica toxin. These results imply that cell surface sialoglyco-proteins serve as receptors for the amoebic toxin.

The polyamine content of Entamoeba was measured by a procedure that involved benzoylation followed by high performance liquid chromatography (h.p.l.c.). A high concentration of putrescine and significant amounts of spermidine and spermine were found in actively growing trophozoites and in the cyst forms of the organism. In contrast, trophozoites in stationary phase had greatly reduced amounts of putrescine and exhibited peaks in h.p.l.c., possibly indicative of acetylated polyamines. αD,Ldifluoromethylornithine (DFMO) lowered the concentration of polyamines in growing trophozoites, but did not inhibit the degree of proliferation. There is evidence for pathways of polyamine biosynthesis in Entamoeba other than through ornithine decarboxylase (ODC).

Pathogenic Entamoeba histolytica isolated from patients with clinical amoebiasis can be differentiated from nonpathogenic E. histolytica obtained from asymptomatic carriers on the basis of the electrophoretic pattern of their isoenzymes. Virulence of different strains of axenically grown trophozoites of Entamoeba histolytica, as determined by various laboratory tests, such as damage to tissue culture monolayers, or their ability to cause an hepatic abscess in a hamster, are known to vary considerably. Reassociation of trophozoites of strain HK-9 with certain Escherichia coli strains for short periods of time markedly augmented their virulence, as tested by the above-mentioned methods. The bacterial association, however, did not cause any change in the electrophoretic pattern of amoebic isoenzymes (zymodeme).

Guinea pig colonic epithelial cells release a soluble lectin capable of agglutinating numerous strains of Shigella and Escherichia coli as well as other bacteria. Using pure oligosaccharides and glycopeptides with well-defined structures to inhibit the agglutination of Shigella flexneri 1b by the soluble intestinal lectin, we have been able to demonstrate that the latter recognises different structural types. Inhibition by human milk glucoprotein glycopeptides with biantennary glycans of the N-acetyllactosamine type was dependent on the simultaneous presence of unsubstituted terminal non-reducing galactose residues and of a fucose residue α-1,6-linked to the asparagine-conjugated N-acetylglucosamine residue. Unsubstituted terminal non-reducing galactose was also determinant for inhibition by human milk oligosaccharides. Finally oligosaccharides possessing the Man (α1-2) Man structure inhibited more effectively than those with a Man(α1-3)Man sequence. The fact that these different structural motifs were all inhibitory raises the problem of the possible existence of a multispecific lectin or of several different lectins in the guinea pig colonic mucosa mediating bacterial adherence.

The different cell forms in the life cycle of Entamoeba invadens (trophozoites, precysts, and cysts) were rapidly and quantitatively separated on density step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll). With this method, the gradual process of encystation by E. invadens trophozoites could be monitored. Percoll gradients were also efficient in separating trophozoites of Entamoeba histolytica and bacteria. After purification on Percoll, trophozoites display no evidence of damage when examined by light microscopy and no loss in viability as judged by their ability to multiply.

Axenically grown pathogenic and non-pathogenic isolates of Entamoeba histolytica have been shown to adhere to mammalian epithelial cells and bacteria by virtue of carbohydrate-binding proteins present on their cell surfaces. The interaction of amoeba isolates of low pathogenicity with a variety of gram-negative bacteria, mainly Escherichia coli strains which are readily ingested by the amoebae after relatively short periods, significantly increased the ability of the trophozoites to: (a) destroy and ingest intestinal epithelial cells; (b) secrete a cytopathic substance which morphologically affects a variety of tissue-cultured cells; and (c) cause hepatic abscesses in hamsters. Addition of carbohydrates that inhibit the lectin-mediated attachment of bacteria to amoebae prevented the enhancement of virulence. Interaction of the amoebae with bacteria that were heat-inactivated, glutaraldehyde-fixed or disrupted by sonication, as well as with bacteria precoated with antibodies or concanavalin A, did not lead to an increase in virulence. Moreover, short prior treatments of the bacteria with inhibitors of protein synthesis, but not with cell-wall synthesis inhibitors, also prevented the stimulation. The results indicate that interactions of amoebae with certain bacteria may be responsible for the increase in amoebic virulence.

Carbohydrate-binding activity present on the Entamoeba histolytica cell surfaces was found to mediate the adherence of two types of bacteria, Escherichia coli serotype 055 and Salmonella greenside 050. Adherence was inhibited by low-molecular-weight carbohydrates (10 mg/ml) such as galactose, lactose, and N-acetylgalactosamine, as well as by asialofetuin and the lipopolysaccharide extracted from E. coli 055. Mild periodate oxidation of the bacteria inhibited their adherence, whereas heat inactivation, glutaraldehyde fixation, or γ-irradiation had no effect. On the other hand, pretreatment of trophozoites with glutaraldehyde, cytochalasin B, or cold (5°C) abolished adherence. None of these treatments, however, affected the attachment of bacteria that contain on their cell surface type I pili with mannose-binding capacity. These findings lend further support to our earlier observations on how amoebae interact with bacteria.

This chapter contains section titled: Experimental System, Immune Responses in SelfLimiting Infections, Immunodepression in SelfLimiting Infections, Immune Response in Non SelfLimiting Infections

(Mirelman) We still dont know enough about host effectors and microenvironments and how are they controlled. Changes in the host could modulate the expression of the parasites genome under conditions that are controlled by the hosts general state-for example, bacterial flora, nutrition, secondary infections, and intake of other medications. These factors may facilitate the development, in vivo in the human host, of a parasitic virulence that would not otherwise have developed under normal conditions. We must try to understand how the parasite may be affected by these varying conditions.

Discussion of this chapter on uptake and fate in normal macrophages the knowledge base for nitrogen input activation and intracellular killing in mouse and human mononuclear phagocytes surface antigenic determinants on different developmental stages of t. cruzi, and their possible role in evasion mechanisms

Discussion of leishmanial excreted factors and strain specificity chemistry of excreted factors production of excreted factors and the leishmanial cell surface possible biological role of leishmanial excreted products

Discussion of definitions and assay systems

This chapter contains section titled: Nucleotide Exchange, Purine and Pyrimidine Synthesis, The Role of HostCell Protein Synthesis, Antigenic Mutants

Cysts of Entamoeba invadens obtained under axenic culture conditions have been reported to be similar to cysts of the human intestinal parasite E histolytica both in morphology and chitin presence in their wails. Mature E. invadens cyst forms, isolated from cultures following discontinuous Percoll gradient sedimentation were resistant (>80%) to detergent treatment. Addition of chitin synthesis inhibitors such as Polyoxin D and Nikkomycin (50 μg/ml) to cultures in encystation media markedly inhibited (>85%) the formation of detergent resistant cysts and prevented the incorporation of radiolabeled chitin precursor N-acetyl[³H]glucosamine. These findings suggest that chitin synthesis inhibitors may serve as drugs which specifically block the life cycle of the Entamoeba parasite.

A method for preparing an isolated colonic loop (Thiry-Vella) in a living rabbit is described. The loop, with its intact neurovascular supply, continues to secrete clear colonic mucus for more than 2 months. The chemical composition of the mucus, collected daily for 2 months, was analyzed and shown to be a high molecular weight glycoprotein composed of approximately equimolar amounts of protein and carbohydrate. The main sugars found were N-acetylgalactosamine, N-acetylglucosamine, galactose, and sialic acid. The most prominent amino acids were threonine, aspartic acid, glycine and serine. A considerable flattening and athrophy of the glandular structure of the isolated colonic loop was observed during the 2 months. This fact did not markedly affect the amount and chemical composition of the mucus that was collected daily from this loop. This model can be used in vivo to investigate colonic mucus in normal and diseased animals, or even following the administration of various drugs.

Bacterial adherence to animal cell surfaces is interesting not only because of its relation to pathogenicity, but also because its studies provide insight into determinants of intercellular recognition. In order to colonize human mucosal surfaces, bacteria have to bind first to the epithelial cell surface, otherwise they will be discarded by host defense mechanisms. In many gram negative organisms, surface pili (adhesins) mediate their binding to epithelial cells. The adherence of numerous types of these organisms to a variety of eukaryotic cells (epithelial, phagocytes, yeasts or red blood cells) is mediated by type I pili that exhibit manonose binding activity. Pili polypeptide subunits obtained after dissociation with saturated guanidine HCl partially retained their mannose binding capacity. Mannose binding adhesins have also been found to be associated with the flagella of certain Escherichia coli and Serratia marcescens strains or with outer membranes of E. coli. The nature of the mannose containing glycoprotein receptors on the animal cells is still unknown. There is good reason to believe that mannose-specific attachment plays a role in infectivity as the administration of mannose derivatives was effective in diminishing experimental E. coli urinary tract infection in mice and certain types of diarrhea. Other types of bacterial pili known to mediate mannose-insensitive adherence to eukaryotic cells have been reported. Thus E. coli originating from human urinary tract infection have an affinity for glycolipids of the globoside series, and enterotoxicogenic strains having a different array of organelles which facilitate colonization, display affinity for certain ganglioside, as well as to yet unidentified receptors. Studies on the adherence of non pilated clinical isolates of Shigella flexneri to the colonic epithelial cells of guinea pigs revealed that their attachment could be specfically inhibited by fucose or glucose. However, in contrast to other known bacterial adherence mechanisms, the adhesin, that mediates the attachment of the bacteria to the mucosal surface, was detected not on the Shigella surface but as an intestial proteinous component that is released together with the mucus gel by the colonic epithelial cells. The intestinal adhesin binds to certain sugars of the bacterial lipopolysaccharide which serve as receptors, and it causes the agglutination of Shigella and other types of bacteria.

Guineapig colonic epithelial cells released by treating sections of the colon with solutions containing EDTA, dithiothreitol, and citrate avidly adhered Shigella flexneri bacteria. Separation of the intestinal cells from nonbound bacteria was achieved by differential sedimentation on a Percoll gradient. Adherence of S. flexneri to the colonic cells was Ca²⁺ (1 mM) and time dependent. The pH optimum was pH 6.2, and almost no attachment (

Entamoeba histolytica trophozoites were found to be very selective in their interactions with bacteria. Two principal mechanisms were shown to be responsible for these interactions. Certain bacteria, such as a number of Escherichia coli and Serratia marcescens strains which are known to contain mannose-binding components on their cell surface, bound to mannose receptors on the amoeba membrane. This attachment was markedly inhibited by α-methylmannoside (0.5%), especially when the incubations were done at low temperature (5°C). Other bacterial species, such as Shigella flexneri and Staphylococcus aureus, which does not possess a mannose-binding capacity, attached to the amoebae, but only with the aid of concanavalin A or after opsonization of the bacteria with immune serum. In both types of attachment, between 40 and 100 bacterial bound per amoeba, and considerable ingestion of bacteria into amoeba vacuoles was observed at 37°C. The attachment of opsonized bacteria to the amoebia does not appear to be mediated by Fc receptors since Fab' dimers obtained after pepsin digestion of immunoglobulin were capable of mediating adherence. Furthermore, preincubation of the amoebae with aggregated human immunoglobulin G or with heat-inactivated immune serum and EDTA did not inhibit the attachment of opsonized bacteria. The attachment of opsonized bacteria was markedly inhibited by N-acetylglucosamine-containing glycoconjugates, such as peptidoglycan and chitin oligosaccharides, as well as by N-acetylgalactosamine. These results indicate that amoebae can attach and ingest bacteria either by using their membrane-associated carbohydrate-binding protein or by having their mannose-containing cell surface components serve as receptors.

Trophozoites of Entamoeba histolytica adhere to and phagocytize red blood cells and bacteria. Furthermore, in the initial step of the amoebic infectious process the parasite attaches to intestinal epithelial cells. A lectin (carbohydrate-binding protein) which apparently has a role in the attachment of the parasite to host cells was found in trophozoites of E. histolytica. When amoeba cells were disrupted by freeze-thawing, the lectin activity, as determined by haemagglutination of human erythrocytes, remained associated with the sedimented membrane fraction. This activity was pH dependent and heat and oxidation-sensitive, and was destroyed by proteolysis and on autoincubation. Moreover, the lectin activity was inhibited by a variety of N-acetylglucosamine-containing compounds such as chitin and chitin oligosaccharides, bacterial peptidoglycan, rabbit colonic mucus, bovine and human serum, an IgA fraction isolated from human colostrum, and IgG from sera of amoebiasis patients. These glycoconjugates also interfered with the adherence of intact radiolabelled amoeba trophozoites to human intestinal epithelial cells as well as their attachment to red blood cells. Although the lectin activity and the toxin-like activity previously found in E. histolytica seem to be two separate substances, they share a number of properties which suggest that they are related and may have a function in pathogenicity.

A new method for preparing an isolated colonic loop in a living rabbit is described. The loop with its intact neurovascular supply can be used as as a "living test tube" to study the adherence of microorganisms to intestinal mucosa. Moreover, the clear colonic mucus produced by the loop can be used to study its physiochemical nature and protecting properties in health and disease.

Surgical sutures are known to potentiate the development of wound infection. The purpose of this study was to investigate whether the capability of bacteria to adhere to various types of sutures has a significant effect on their ability to cause infections. Bacterial adherence to sutures was quantitatively measured using radiolabeled bacteria. In vitro adherence assays revealed remarkable variations in the affinity of bacteria to the various sutures: nylon bound the least bacterria while bacterial adherence to braided sutures (silk, Ti-cron, Dexon) was five to eight folds higher. The degree of infection obtained in mice in the presence of different sutures nicely correlated with their adherence properties. The different removal rate of adherent bacteria (glutaraldehyde-fixed) from various sutures by the tissue factors in mice supports the hypothesis that bacterial adherence to suture materials plays a significant role in the induction of surgical infection. Our observation points out at the need for careful sutures selection in contaminated wounds. The adherence properties of sutures should be considered in any future surgical suture design.

A comparison was made between properties of a recently discovered Entamoeba histolytica lectin which has a carbohydrate specificity for N-acetylglucosamine oilgosaccharides and the previously found toxin-like principle of the ameba. A separation between these two activities was achieved upon subcellular fractionation by speed centrifugation of freeze-thawed disrupted E. histolytica trophozoites (strain HM-1). Practically all of this lectin activity, as determined by hemagglutination of glutaraldehyde-fixed human erythrocytes, was found associated with the sedimented membrane fraction. This fraction did not affect monolayers of tissue-cultured mammalian cells. On the other hand, the soluble supernatant solution caused extensive damage to the tissue-cultured cells (change in morphology and detachment of cells). Both the lectin and toxin activities were heat-labile and their activities were preserved by the presence of reducing agents and proteolytic enzyme inhibitors. In contrast to the toxin, the isolated lectin was inactive at pH 7.2 and active only at pH 5.7-6.0. Both the lectin and toxin were inhibited by a number of macromolecular compounds such as chitin, peptidoglycan, bovine serum and an IgA fraction isolated from human colostrum. Only the lectin activity, however, was inhibited by low molecular weight chitin oligosaccharides (GlcNAc)n=_2-6 or by lysozyme-digested peptidoglycan subunits. Moreover, fetuin and a ganglioside mixture extracted from ox brain were found to inhibit only the toxin-like activity. The IgG fraction of sera from patients with invasive amebiasis neutralized both lectin and toxin-like activities, while IgG from normal sera failed to neutralize either activity. Although our results indicate that in E. histolytica, lectin and toxin are two separate activities, both of them share a considerable number of properties which does not exclude the possibility that they may be related.

Antibiotic TA inhibited incorporation of diaminopimelic acid and uridine diphosphate-N-acetylglucosamine into Escherichia coli cell walls without altering the ratio of cross-linked to uncross-linked peptidoglycan. Formation of the lipid intermediate was not blocked by TA, suggesting that TA interferes with polymerization of the lipid-disaccharide-pentapeptide.

The enzymatic reactions (transpeptidases) that catalyze the attachment of newly synthesized peptidoglycan to the preexisting cell wall sacculus of both Escherichia coli and P. aeruginosa have been shown to be very sensitive to most β-lactam antibiotics. Biosynthetic studies carried out with a clinical isolate of P. aeruginosa resistant to carbenicillin and cefsulodin showed that the in vitro reactions were also insensitive to most β-lactam antibiotics (up to 50 μg/ml) and only cefotaxime or its tetrazolyl analog, compound LY 97962, had an inhibitory effect at 0.01 μg/ml. The pattern of β-lactam binding proteins obtained upon exposure of intact or presonicated cells to radioactively labeled compound LY 97962 or penicillin G indicates that: intact cells of the clinical isolate are 10 to 50 times less permeable to the antibiotics than is the wild-type strain X-48; β-lactam binding proteins Ia, Ib, and III of the clinical isolate showed poor affinity for penicillin G and cefsulodin, but were similar to the wild type in their affinity for cefotaxime and compound LY 97962. The 2 strains also differed in several of their outer membrane components. These results suggest that the insusceptibility of this clinical isolate is due to a combination of outer membrane impermeability and intrinsic insensitivity to most of the β-lactams on the part of the enzymes which catalyze expansion and growth of peptidoglycan.

The effect of amino acid deprivation on the activities of D-alanine carboxypeptidase (CPase) and peptidoglycan transpeptidase in E. coli was determined. Enzymes were assayed in ether-treated bacteria (ETB) which were permeable to peptidoglycan nucleotide precursors. ETB were prepared at intervals from cultures grown in the presence and absence of a required amino acid. The specific activity of CPase in ETB decreased 50 to 85% during amino acid deprivation. This was paralleled by a 60 to 70% decrease in the specific activity of peptidoglycan transpeptidase. Both enzymes reached their lowest level of activity about 40 min after the onset of amino acid deprivation. The decrease in CPase activity apparently was not due to degradation of the enzyme, since full activity was restored after disruption of ETB by sonication. A decrease in CPase activity was associated with an enhancement of transpeptidation. The peptidoglycan synthesized in vitro by amino acid-deprived ETB was 1.7 times more cross-linked than the peptidoglycan synthesized by control ETB. These results support the proposal that CPase may be involved in regulating transpeptidation in E. coli.

wo homologous strains of Escherichia coli, one of which completely lacked the cell envelope Braun's lipoprotein, were compared with respect to their peptidoglycan synthesis and assembly. Both strains were auxotrophic for diaminopimelic acid and their uptake of radiolabeled diaminopimelic acid was comparable. Analysis of subcellular fractions obtained after mechanical disruption of the cells in a French pressure cell and sedimentation of the cell envelopes showed the existence of a soluble, chromatographically immobile macromolecular peptidoglycan. This labeled peptidoglycan contained a reduced degree of peptide side chain cross-linkages (19 mol % of labeled residues as compared to that present in the insoluble cell sacculus, 27 mol %). In addition, approximately 20% of its peptide side chains terminated in pentapeptide structures versus 1 to 4% in the sacculus. Furthermore, the soluble peptidoglycan of the parent strain also contained covalently bound lipoprotein (4.6%). Extraction of the cell envelope fraction with detergents afforded an additional amount of soluble peptidoglycan. This material was quite similar, in its degree of cross-linkage and amount of covalently bound lipoprotein, to the peptidoglycan present in the detergent-insoluble sacculus. These results indicate that peptidoglycan strands which are, in part, covalently linked to lipoprotein are late stage synthesis intermediates which subsequently become covalently attached to the preexisting sacculus.

A lectin (carbohydrate binding protein) has been found in extracts of a number of axenically grown trophozoites of Entamoeba histolytica strains. The strains grown in Diamond's TYI-S-33 media were HK-9, 200:NIH and HM-1: IMSS. Strain HU-1: MUSC (HSC) was grown monoxenically in the same medium. The amoeba lectin agglutinates glutaraldehyde-fixed red blood cells. This activity is pH dependent, heat and oxidation sensitive and is destroyed by proteolysis upon auto-incubation. The relative agglutinating potency of the different strains was investigated. Strain HSC had the highest specific activity (210 units/mg protein) and strain HM-1 the lowest (14 units/mg). One unit of haemagglutinating activity is defined as the amount of leetin present in 1 ml of extract which will agglutinate 1 ml of 4 per cent red blood cells. Upon subcellular fractionation of the lectin present in extracts of strain HK-9, two thirds of the activity were detected in the soluble, non-sedimentable (100,000 x g, 60 min) fraction. Partial hydrolysate of chitin was found to inhibit the haemagglutinating activity. Among the oligosaccharides of N-acetylglucosamine, the trimer and tetramer were the most potent inhibitors. The lectin was purified approximately 300 fold by a one step affinity chromatography on a chitin column. The loading and elution from the column were based on the pH dependence of the lectin activity.

Recently, it was suggested that a mannose-specific lectin on the bacterial cell surface is responsible for the recognition by phagocytic cells of certain nonopsonized Escherichia coli strains. In this study we assessed the interaction of two strains of E. coli at different phases of growth with a monolayer of mouse peritoneal macrophages and developed a direct method with [¹⁴C]mannan to quantitate the bacterial mannose-binding activity. Normal-sized bacteria were obtained from logarithmic and stationary phases of growth. Nonseptated filamentous cells were formed by growing the organisms in the presence of cephalexin or at a restrictive temperature. Attachment to macrophages of all bacterial forms was inhibited by methyl α-D-mannoside and mannan but not by other sugars tested. The attachment of stationary phase and filamentous bacteria to macrophages, as well as their mannose-binding activity, was similar, whereas in the exponential-phase bacteria they were markedly reduced. The results show a linear relation between the two parameters (R=0.98, P

A total of 393 clinical bacterial isolates were tested for their ability to agglutinate yeast cells of either Saccharomyces cerevisiae or Candida albicans. A positive agglutination of yeasts that could be prevented by methyl α-D-mannoside was taken as an indication for the possible presence of a mannose-specific lectin (carbohydrate-binding protein) on the surface of the tested bacteria. Agglutination tests on glass slides showed that 38% of all the isolates tested were positive in their capacity to agglutinate yeasts. Among the various strains tested, all isolates of Serratia marcescens, Proteus morganii, and Citrobacter diversus, as well as 94% of Klebsiella pneumoniae, were positive. On the other hand, only 46% of the Escherichia coli, 48% of the salmonellae, 44% of the Citrobacter freundii, and 71% of the Aeromonas hydrophila isolates were positive. A quantitative determination of the lectin activity done by observing the agglutination of yeasts in microtiter plates showed that S. marcescens isolates were the most avid binders of the yeast, whereas Klebsiella and Citrobacter isolates were the weakest.

The intrinsic effect of three novel methoxyimino derivatives of cephalosporin (cefotaxime [syn HR 756]; its anti isomer, R 02 5328 A; and the syn S-oxide derivative, HR 109) on the biosynthesis of peptidoglycan of Pseudomonas aeruginosa X-48 was investigated. Cefotaxime at very low concentrations (50% inhibition at 0.025 μg/ml) inhibited the transpeptidase reaction which catalyzes the incorporation and attachment of newly synthesized peptidoglycan to the preexisting cell wall sacculus. The S-oxide compound, HR 109, was a much less efficient inhibitor of this reaction (50% inhibition at 0.55 μg/ml), whereas the anti isomer of cefotaxime, R 02 5328 A, had no inhibitory effect. All three compounds were quite similar in being relatively poor inhibitors of D-alanine carboxypeptidase.

A lectin (carbohydrate-binding protein) has been found in extracts of a number of axenically grown trophozoites of Entamoeba histolytica strains. The strains grown in TYI-S-33 medium (Diamond et al., Trans. R. Soc. Trop. Med. Hyg. 72: 431-432, 1978) were HK-9, 200:NIH and HM:IMSS. Strain HU-1:MUSC (HSC) was grown monoxenically in the same medium. The amoebic lectin agglutinated glutaraldehyde-fixed erythrocytes. This activity was pH dependent and heat and oxidation sensitive, and was destroyed by proteolysis upon autoincubation. The relative agglutinating potency of the different strains of amoebae was investigated. Strain HSC had the highest specific activity (210 U/mg of protein), and strain HM-1 had the lowest (14 U/mg). One unit of hemagglutinating activity is defined as the amount of lectin present in 1 ml of extract which will agglutinate 1 ml of 4% erythrocytes. Upon subcellular fractionation of the lectin present in extracts of strain HK-9, two-thirds of the activity was detected in the soluble, nonsedimentable (100,000 x g, 60 min) fraction. Partial hydrolysis of chitin was found to inhibit the hemagglutinating activity. Among the oligosaccharides of N-acetylglucosamine, the trimer and tetramer were the most potent inhibitors. The lectin was purified approximately 300-fold by a one-step affinity chromatography on a chitin column. The loading and elution from the column were based on the pH dependence of the lectin activity.

Methyl a-D-mannopyranoside (uMM), a competitor inhibitor of the binding of mannose by Escherichia coli, was tested for its ability to prevent infection of the urinary tract of mice with infective strains of the organisms. Injection of the bacteria in the presence of the drug resulted in a considerable reduction in the number of bacteriuric mice. In this system «MM was inactive against Proteus mirabilis in accordance with its inability to inhibit the adherence of this organism to epithelial cells in vitro, and methyl a-D-glucopyranoside proved inactive against both E. coli and P. mirabilis.

Ethertreated cells of pseudomonas aeruginosa catalyze the formation of crosslinked peptidoglycan from the two nucleotide precursors uridinediphosphoNacetylglucosamine and uridinediphosphoNacetylmuramyllalanyldγglutamylmeso diaminopimelyldalanyldalanine. The main enzymatic reactions of biosynthesis were similar to those found in Escherichia coli. Part of the reaction products were soluble in 4% sodium dodecylsulfate whereas the other part was covalently bound to the preexisting cell wall peptidoglycan sacculus. The incorporation into cell wall is carried out by a transpeptidation reaction in which the nascent peptidoglycan functions mainly as the donor and the preexisting one as acceptor. The detergentsoluble peptidoglycan is composed of partially crosslinked peptidoglycan strands as well as lowmolecularweight peptidoglycan fragments. Pulsechase biosynthesis experiments show that the detergentsoluble peptidoglycan is an intermediate that eventually becomes covalently bound to the wall. The ddcarboxypeptidase activity of P. aeruginosa is membranebound and does not hydrolyse Cterminal dalanine residues from the llysinecontaining nucleotideprecursor analogue. An ldcarboxypeptidase was also detected in P. aeruginosa.

The intrinsic effect of various βlactam antibiotics on the biosynthesis of peptidoglycan of Pseudomonas aeruginosa X48 was investigated. Most of the cephalosporins and penicillins tested already at 0.5 μg/ml strongly inhibited (a) the incorporation of nascent peptidoglycan into the detergentinsoluble fraction (> 75%), (b) the formation of peptide crosslinkages (> 60%) and (c) the activity of the DDcarboxypeptidase and partially that of the transpeptidase (∼ 90% and ∼ 40% respectively). Another group of βlactam drugs did not inhibit incorporation into the material insoluble in sodium dodecylsulfate, the formation of peptide crosslinkages nor transpeptidase activity. They only partially inhibited the activity of the DDcarboxypeptidaseendopeptidase system (4050% at 0.5 μg/ml). The results obtained differ from those of Presslitz and Ray [Antimicrob. Agents Chemother. 7, 578581 (1975)] and show some resemblance to the effects of βlactams on the biosynthesis of Escherichia coli peptidoglycan.

Oxidative deamination of the epsilon-amino group of lysyl residues to form allysine is the initial reaction in the cross-linking of collagen and elastin in vertebrates. The allysyl residues, generated by lysyl oxidase in this reaction, condense with either other allysyl residues or epsilon-amino groups of lysyl or hydroxylysyl to form aldol or Schiff base cross-links. This paper presents evidence that similar allysyl residues and Schiff base cross-links are synthesized in cell envelopes of Escherichia coli. Acid hydrolysis followed by amino acid analysis of envelopes either reduced with NaB[3H]4 or labeled with [14C]lysine and reduced with NaBH4 yielded allysine and two labeled fragments with elution profiles and molecular weights (250 and 330) consistent with Schiff base products derived at least in part from allysine. When [6-3H]lysine-labeled cell envelopes were incubated at 37 degrees C, gradual release of tritiated water occurred. This suggests that an enzymatic reaction catalyzes the deamination of lysine in E. coli membranes and that the higher molecular weight proteins detected in stationary phase or in log phase cell envelopes after NaBH4 reduction occur as a result of formation of Schiff base cross-links.

A high molecular weight protein aggregate, which agglutinates yeast cells, human epithelial cells and mouse lymphocytes, was isolated from extracts of Escherichia coli by differential centrifugation and gel filtration. The agglutination is specifically inhibited by d-mannose and its derivatives, the best inhibitor being p-nitrophenyl α-d-mannoside. Sodium dodecyl sulfate gel electrophoresis showed that the lectin consists of protein subunits with identical M_r of ∼36500. The amino acid composition of the purified lectin is different from that reported for the type I pili protein, the K99 antigen and the major outer membrane protein Ia of E. coli. The protein appears to be located on the bacterial surface, and is probably involved in the mannose-specific adherence of E. coli to eukaryotic cells.

Antibodies elicited by a synthetic immunogen, (Glu⁶⁰ Ala⁴⁰)_n [AladGlu(LysdAladAla)]^5.1, related to a major class of peptidoglycan precursors, were purified by an affinity column of Sepharose 4B, to which the hapten, AladGlu(LysdAladAla), was covalently attached and were then coupled to Sepharose 4B. Lowmolecularweight [¹⁴C]alaninelabeled products secreted by Micrococcus luteus cells grown in the presence of penicillin G were applied to the antibodycoupled gel. The bound material, which should be univalent to the antibody, was eluted completely by 0.02 mM αtertbutyloxycarbonyllysyldalanyldalanine. Highmolecularweight uncrosslinked soluble peptidoglycan secreted by M. luteus cells grown in the presence of penicillin G was completely bound to the affinity column, regardless of whether the soluble peptidoglycan was labeled in its alanine moieties or in its glucosamine and muramic acid residues. A minor fraction of the bound soluble peptidoglycan was released with 0.02 mM tripeptide (paucivalent fraction). The remainder of the bound material was eluted with 2 mM tripeptide, indicating that a major portion of the highmolecularweight material was multivalent. When glycanlabeled soluble peptidoglycan was stored in buffered saline at 5°C for several months, a fraction of the labeled material was no longer bound to the affinity gel. Digestion of this nonbinding fraction, as well as the paucivalent and multivalent fractions with hen eggwhite lysozyme, was consistent with the affinity chromatographic data. These results indicate that an amidase present in these preparations cleaved different amounts of peptide from the soluble peptidoglycan preparations, producing strands which are polydisperse with respect to their peptide substitution.

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.

The biosynthesis of the linkage region between peptidoglycan and the ribitol teichoic acid was investigated in the bacteriophage-resistant, teichoic acid-less mutant S. aureus 52A5. Membrane preparations of this strain were found to be incapable of forming the first intermediate of the biosynthetic pathway, namely, the transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to the acceptor molecule, which presumably is undecaprenol phosphate. The addition of heat-inactivated membrane preparations of S. aureus 52A2 (which normally has ribitol teichoic acid) that had been preincubated with UDP-GlcNAc to membranes of strain 52A5 enabled the synthesis of teichoic acid. These data suggest that the mutational defect in the teichoic acid-less organism is in the synthesis of the first compound of the linkage unit, and this is apparently the reason for its absence in the cell walls.

The last stages of murein biosynthesis were studied in relation to the division cycle of E. coli in cells synchronized by amino acid starvation. Murein synthesis and the activities of the D-alanine carboxypeptidase and transpeptidase were found to vary significantly during the cell cycle. Maximal synthesis and transpeptidation were observed immediately after cell division, whereas maximal D-alanine carboxypeptidase activity was detected before cell division. These results are in agreement with earlier findings that before cell division there is a stage of increased hydrolysis of the C-terminal D-alanine moiety of newly synthesized murein strands.

The attachment of Escherichia coli and Salmonella typhi to mouse peritoneal macrophages was inhibited by D-mannose, methyl α-D-mannopyranoside and yeast mannan, but not by any other sugar tested. D-Mannose and its derivatives also inhibited the attachment of E. coli to human polymorphonuclear leucocytes. Mannan inhibited phagocytosis when preincubated with E. coli, but not when preincubated with leucocytes. Attachment of opsonized bacteria to leucocytes was not inhibited by D-mannose or methyl α-D-mannopyranoside nor by any other sugar tested. Our results suggest that the surface of phagocytes, like that of epithelial cells, contains D-mannose residues which serve for the attachment of certain Gram negative bacteria.

Digestion by five different lysozymes (mucopeptide N-acetylmuramoylhydrolases, EC 3.2.1.17) of a soluble uncross-linked peptidoglycan secreted by Micrococcus luteus (Mirelman, D., Bracha, R. and Sharon N. (1974) Biochemistry 13, 5045-5053) was investigated. Hen egg-white and human lysozymes converted the peptidoglycan into the disaccharide GlcNAc-β(1 → 4)-N-acetylmuramic acid (-MurNAc), the tetrasaccharide (GlNAc-β(1 → 4)-MurNAc)₂ and the disaccharide-hexapeptide: {A figure is presented} With goose egg-white lysozyme, mainly the disaccharide and the disaccharidehexapeptide were produced, in approximately equimolar amounts. Papaya lysozyme afforded mainly products without peptide substitution, such as the disaccharide and tetrasaccharide, while T4 phage lysozyme gave only the disaccharide-hexapeptide. Lysozymes from different sources thus differ in their specificity requirements with respect to peptide substitution on the lactyl moiety of N-acetylmuramic acid. This pattern of specificity is discussed in terms which correlate it with the differences in the structures of their presumed natural substrates. We postulate that evolutionary changes in the substrate structure may have influenced the development of the active site of lysozyme so that it could function most efficiently with the particular natural substrate encountered by each species.

Both the β lactam antibiotic, cephalexin, and the deoxyribonucleic acid synthesis inhibitor, nalidixic acid, are known to inhibit cell division in E. coli and induce the formation of filaments. The biosynthesis of murein was investigated in these filaments and compared with the murein synthesized by the normally dividing rods of E. coli PAT 84. Differences were found in the extent of peptide side chain cross linkage. Filamentous cells had higher extents of cross linkages in their newly synthesized murein. Quantitative analyses of the D alanine carboxypeptidase and transpeptidase reactions in the different cells revealed that the carboxypeptidase activity of the filamentous cells was partially inhibited. These results were similar to those previously found with filaments that were obtained after growth of the thermosensitive division mutant at its restrictive temperature. It is concluded that the formation of new cell ends (septa) depends on the proper balance between the activities of the D alanine carboxypeptidase that regulates the availability of precursor donors and the transpeptidase, which catalyzes cross linking and attachment of newly synthesized murein.

The ability of human milk galactosyltransferase to attach dgalactose residues quantitatively to the C4 of Nacetylglucosamine moieties at the ends of oligosaccharides has been utilized for the specific labeling and quantitative determination of the chain length of the glycan moiety of the bacterial cell wall. The average polysaccharide chain length of the soluble, uncrosslinked peptidoglycan secreted by Micrococcus luteus cells on incubation with penicillin G was studied with this technique and found to be approximately 70 hexosamines long. Furthermore, the peptidoglycan chain length of Escherichia coli sacculi of different cell shapes and dimensions was determined both in rodshaped cells and in filaments induced by temperature shift of a division mutant or by addition of cephalexin or nalidixic acid. The average chain length found in most of these sacculi was between 70 and 100 hexosamines long. Small spherical mini cells had chain lengths similar to those of the isogenic rodlike cells.

A series of bacterial cell wall glycopeptides of low molecular weight and cell wall nucleotide precursors have been tested for their inhibitory action on the digestion by T4 lysozyme of a radioactively labeled linear uncrosslinked peptidoglycan. The disaccharidepeptides GlcNAcMurNAclAladGlu(A₂pm) (C₅) and GlcNAcMurNAclAladGlu(A₂pmdAla) (C₆) as well as the monosaccharidepeptide MurNAclAladGlu(A₂pm) were found to be good competitive inhibitors (with similar K₁ values) whereas the disaccharidepentapeptide GIcNAcMurNAclAlad GluGly lLysdAla was a poor inhibitor. T4 lysozyme did not catalyse transglycosylation reactions from Escherichia coli B peptidoglycan to the disaccharidepeptide C₆. No changes were seen in the circular dichroism spectra (200250 nm) or fluorescence emission spectra upon binding of the good inhibitors. The results obtained indicate that T4 lysozyme has a small active site capable of recognizing a unit consisting of MurNAclAladGlu(A₂pm).

Peptidoglycan biosynthesis during a bacterial division cycle was investigated in the thermosensitive division mutant Escherichia coli PAT 84. Synchronous cell division of this organism was initiated by a shift down from restrictive to permissive growth temperature. Cells harvested at different times after a shift down of temperature served as representatives of the various stages during cell division. These cells were made permeable to peptidoglycan nucleotide-sugar precursors by pretreatment with ether and were found capable of catalyzing the polymerization of externally added precursors as well as the covalent attachment of the newly synthesized peptidoglycan strands to those preexisting in the cell wall. Differences were observed in the rate of peptidoglycan synthesis and in the extent of peptide side-chain cross-linkage at the various stages of division. Nonseptate filaments, formed at the restrictive temperature, incorporated significantly more peptidoglycan which was more cross-linked than in normally dividing cells grown at the permissive temperature. Quantitative analyses of the carboxypeptidase and transpeptidase reactions in cells at different stages of division were performed and the inhibitory effect of a number of β-lactam antibiotics was investigated. Of special significance was the finding that low doses of penicillin or growth at restrictive temperature, which did not affect transpeptidation, partially inhibited the carboxypeptidase activity. This inhibition was paralleled by an increase in incorporation of newly synthesized peptidoglycan into the preexisting cell wall. We therefore propose that carboxypeptidase activity regulates the availability of peptidoglycan precursor(s) for attachment to the preexisting peptidoglycan by transpeptidation.

The two bacterial cell wall peptidoglycan precursors UDP-MurNAc-l-Ala-d-iso Glu-l-Lys-d-Ala-d-Ala and UDP-GlcNAc labeled in their amino sugars with either tritium or carbon-14 accumulated in cells of Micrococcus luteus that were incubated for short periods of time in a minimal medium to which [¹⁴C]glucose or [³H]glucose together with Vancomycin were added. The radioactive nucleotides were extracted from the cells with cold trichloroacetic acid, and their purification was achieved by paper electrophoresis followed by paper chromatography.

Lysozyme from bacteriophage T4 was found to digest a soluble, uncrosslinked peptidoglycan which is secreted by cells of Micrococcus luteus when incubated in the presence of penicillin G. Analysis of the enzymatic degradation products shows that T4 acts as an endoacetylmuramidase capable of cleaving glycosidic bonds only at muramic acid residues that are substituted with peptide sidechains. The results indicate that the secreted peptidoglycan may consist of a mixture of chains, approximately half of which are substituted by peptide side chains on most of their muramic acid residues, while the other half is made up of chains in which the muramic acid moieties are unsubstituted.

Wheat germ agglutinin was found to agglutinate cells of Escherichia coli PAT 84, Micrococcus luteus, Staphylococcus aureus H, and of S. aureus 52A5, but not cells of S. aureus 52A2. Interaction of wheat germ agglutinin with a soluble linear peptidoglycan secreted by Micrococcus luteus and with the teichoic acid of S. aureus H was demonstrated by agar gel diffusion, quantitative precipitation and inhibition of hemagglutination of trypsinized rabbit erythrocytes. No interaction could be demonstrated with the teichoic acid from a phageresistant mutant (S. aureus 52A2) which lacks Nacetyldglucosamine residues. All interactions were specifically inhibited by low concentrations of chitotriose (GlcNAcβ1 → 4GlcNAcβ1 → 4GlcNAc) and the bacterial cell wall tetrasaccharide, GlcNAcβ1 → 4MurNAcβ1 → 4GlcNAcβ1 → 4MurNAc. Hemagglutinationinhibition experiments showed that the linear peptidoglycan and the teichoic acid of S. aureus H were several thousand times more potent inhibitors of wheat germ agglutinin than was Nacetyldglucosamine. Comparison of the efficiency of different saccharides in inhibition of hemagglutination and precipitation of polymers by wheat germ agglutinin, strongly suggests that secondary, nonspecific interactions contribute to the binding of the lectin to the polymers.

The purification and properties of a novel type of murein transglycosylase from E. coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low molecular weight products. The two prominent muropeptide fragments in the digest are the disaccharide tripeptide N acetylglucosamine N acetylmuramic acid L alanine D iso glutamic acid meso diaminopimelic acid and the corresponding disaccharide tetrapeptide N acetylglucosamine N acetylmuramic acid L alanine D iso glutamic acid meso diaminopimelic acid D alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1→6 anhydro linkage. The new lytic enzyme is designated as a murein:murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.

GROWTH of fungal hyphae is the result of a complex and poorly understood process of cell wall synthesis and extension, that is restricted to the hyphal apex^1-3. In fungi with chitin-glucan hyphal walls, such as the Deuteromycetes Trichoderma viride and Fusarium solani¹, hyphal extension and septa formation involve the synthesis of chitin in hyphal tips and septa⁴. We therefore studied the effect on such fungi of wheat germ agglutinin (WGA), a lectin which interacts specifically with chitin oligomers^5,6.

Incubation of Micrococcus luteus cells in the presence of penicillin G leads to accumulation in the culture medium of a linear uncross-linked peptidoglycan. The amount of secreted peptidoglycan was dependent on the concentration of penicillin and paralleled the rate of cell wall synthesis, and the secretion was not accompanied by any cell lysis. Analysis of the peptidoglycan revealed that over 84% of its weight could be accounted for by N-acetylglucosamine, Ar-acetylmuramic acid, Ala, Glu, Gly, and Lys in the molar ratios of 2:2:3:1:1:1. The product was digested by lysozyme to yield mainly the disaccharide GlcNAc-β-(1⟶4)-MurNAc and the disaccharide-hexapeptide GlcNAc-β-(1⟶4)-MurNAc-L-Ala-D-i-Glu(Gly)-L-Lys-D-Aia-D-Ala. The molecular weight of the secreted peptidoglycan, estimated by ultracentrifugation, was found to be 38, 000, corresponding to a chain length of approximately 50 disaccharide units, half of which are substituted by the hexapeptide on their N-acetylmuramic acid residues. A similar value for the molecular weight of the peptidoglycan was obtained from periodate oxidation and β-elimination studies. Muramic acid was found at the reducing end of the peptidoglycan. In addition to the uncross-linked peptidoglycan, penicillin also promoted the secretion of a hexapeptide in equimolar amounts to that of unsubstituted muramic acid residues in the secreted peptidoglycan. It is concluded that the secretion of the peptidoglycan is due to the inhibition by penicillin of the attachment of newly synthesized peptidoglycan strands by transpeptidation to the preexisting cell wall. The secretion of the free hexapeptide is assumed to be the result of the action of a penicillin-insensitive amidase which acts on the newly synthesized peptidoglycan strands.

Cell wall preparations of Staphylococcus aureus catalyse in vitro peptidoglycan synthesis from UDP-GlcNAc, UDP-MurNAc-pentapeptide and glycine without the addition of soluble tRNA and glycyl-tRNA synthetase. Newly synthesised peptidoglycan is partially cross-linked by transpeptidation as evidenced by the release of [¹⁴C]-D-alanine and this reaction is strongly inhibited by small concentrations of penicillin G. This antibiotic also partially inhibits the incorporation of glycine into insoluble cell wall peptidoglycan.