Ken Hirata

Cloning of a virulence factor of Entamoeba histolytica. Pathogenic strains possess a unique cysteine proteinase gene.

Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined.

Cysteine proteinases from distinct cellular compartments are recruited to phagocytic vesicles by Entamoeba histolytica

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence.

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model

ABSTRACT Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

A novel Entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target.

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.

The cathepsin L of Toxoplasma gondii (TgCPL) and its endogenous macromolecular inhibitor, toxostatin.

Toxoplasma gondii is an obligate intracellular parasite of all vertebrates, including man. Successful invasion and replication requires the synchronized release of parasite proteins, many of which require proteolytic processing. Unlike most parasites, T. gondii has a limited number of Clan CA, family C1 cysteine proteinases with one cathepsin B (TgCPB), one cathepsin L (TgCPL) and three cathepsin Cs (TgCPC1, 2, 3). Previously, we characterized toxopain, the only cathepsin B enzyme, which localizes to the rhoptry organelle. Two cathepsin Cs are trafficked through dense granules to the parasitophorous vacuole where they degrade peptides. We now report the cloning, expression, and modeling of the sole cathepsin L gene and the identification of two new endogenous inhibitors. TgCPL differs from human cathepsin L with a pH optimum of 6.5 and its substrate preference for leucine (vs. phenylalanine) in the P2 position. This distinct preference is explained by homology modeling, which reveals a non-canonical aspartic acid (Asp 216) at the base of the predicted active site S2 pocket, which limits substrate access. To further our understanding of the regulation of cathepsins in T. gondii, we identified two genes encoding endogenous cysteine proteinase inhibitors (ICPs or toxostatins), which are active against both TgCPB and TgCPL in the nanomolar range. Over expression of toxostatin-1 significantly decreased overall cysteine proteinase activity in parasite lysates, but had no detectable effect on invasion or intracellular multiplication. These findings provide important insights into the proteolytic cascades of T. gondii and their endogenous control.

Pantropic retroviral vectors mediate gene transfer and expression in Entamoeba histolytica

Transformation of Entamoeba histolytica has been previously reported, but the foreign genes have all been replicated episomally. Pantropic retroviral vectors based on the Moloney murine leukemia virus with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) have an extremely broad host range and can be concentrated to high titer. To investigate whether these pseudotyped, pantropic vectors can mediate gene transfer and expression in E. histolytica, we constructed a retroviral vector, in which a hygromycin phosphotransferase is expressed from the E. histolytica actin promoter. Data confirm the infection, integration, and expression of a foreign gene mediated by the provirus. To our knowledge, this is the most evolutionarily distant example of successful integration and expression of a mammalian retrovirus. Pantropic retroviral vectors may thus facilitate genetic analysis in species lacking transformation systems.

Entamoeba histolytica Induces Intestinal Cathelicidins but Is Resistant to Cathelicidin-Mediated Killing

ABSTRACT The enteric protozoan parasite Entamoeba histolytica is the cause of potentially fatal amebic colitis and liver abscesses. E. histolytica trophozoites colonize the colon, where they induce inflammation, penetrate the mucosa, and disrupt the host immune system. The early establishment of E. histolytica in the colon occurs in the presence of antimicrobial human (LL-37) and murine (CRAMP [cathelin-related antimicrobial peptide]) cathelicidins, essential components of the mammalian innate defense system in the intestine. Studying this early step in the pathogenesis of amebic colitis, we demonstrate that E. histolytica trophozoites or their released proteinases, including cysteine proteinase 1 (EhCP1), induce intestinal cathelicidins in human intestinal epithelial cell lines and in a mouse model of amebic colitis. Despite induction, E. histolytica trophozoites were found to be resistant to killing by these antimicrobial peptides, and LL-37 and CRAMP were rapidly cleaved by released amebic cysteine proteases. The cathelicidin fragments however, did maintain their antimicrobial activity against bacteria. Degradation of intestinal cathelicidins is a novel function of E. histolytica cysteine proteinases in the evasion of the innate immune system in the bowel. Thus, early intestinal epithelial colonization of invasive trophozoites involves a complex interplay in which the ultimate outcome of infection depends in part on the balance between degradation of cathelicidins by amebic released cysteine proteinases and upregulation of proinflammatory mediators which trigger the inflammatory response.

Hsp90 Inhibitors as New Leads to Target Parasitic Diarrheal Diseases

ABSTRACT Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

X-Ray Structures of Thioredoxin and Thioredoxin Reductase from Entamoeba Histolytica and Prevailing Hypothesis of the Mechanism of Auranofin Action.

The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.

4-aminopyridyl-based lead compounds targeting CYP51 prevent spontaneous parasite relapse in a chronic model and improve cardiac pathology in an acute model of Trypanosoma cruzi infection

Background Chagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice. Methodology/Principal findings Both acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart. Conclusions/Significance The positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life.