Universal HIV and Birth Cohort HCV Screening in San Diego Emergency Departments.

Universal HIV and HCV screening in emergency departments (ED) can reach populations who are less likely to get tested otherwise. The objective of this analysis was to evaluate universal opt-out HIV and HCV screening in two EDs in San Diego. HIV screening for persons aged 13-64 years (excluding persons known to be HIV+ or reporting HIV testing within last 12 months) was implemented using a 4th generation HIV antigen/antibody assay; HCV screening was offered to persons born between 1945 and 1965. Over a period of 16 months, 12,575 individuals were tested for HIV, resulting in 33 (0.26%) new HIV diagnoses, of whom 30 (90%) were successfully linked to care. Universal screening also identified 74 out-of-care for >12-months HIV+ individuals of whom 50 (68%) were successfully relinked to care. Over a one-month period, HCV antibody tests were conducted in 905 individuals with a seropositivity rate of 9.9% (90/905); 61 seropositives who were newly identified or never treated for HCV had HCV RNA testing, of which 31 (51%) resulted positive (3.4% of all participants, including 18 newly identified RNA positives representing 2% of all participants), and 13/31 individuals (42%) were linked to care. The rate of newly diagnosed HCV infections exceeded the rate of newly diagnosed HIV infections by >7-fold, underlining the importance of HCV screening in EDs.

Point-of-care diagnosis of invasive aspergillosis in non-neutropenic patients: Aspergillus Galactomannan Lateral Flow Assay versus Aspergillus-specific Lateral Flow Device test in bronchoalveolar lavage.

BACKGROUND: We compared new Aspergillus Galactomannan Lateral Flow Assay with the newly formatted Aspergillus-specific Lateral Flow device tests for the diagnosis of invasive pulmonary aspergillosis (IPA) in non-neutropenic patients. METHODS: We performed both tests in 82 bronchoalveolar lavage fluid samples from 82 patients at risk for IPA but without underlying haematologic malignancy. Samples were collected between September 2016 and September 2018 at the University of California San Diego, United States. IPA was classified following two published consensus criteria. RESULTS: Classification of cases varied widely between the two consensus criteria. When using criteria established for the intensive care unit, 26/82 patients (32%) met criteria for proven or putative IPA. Both point-of-care assays showed sensitivities ranging between 58% and 69%, with specificities between 68% and 75%. Sensitivity increased up to 81% when both tests were combined. CONCLUSION: The study outlines the need for updated, unified and more broadly applicable consensus definitions for classifying IPA in non-neutropenic patients, a work that is currently in progress. Both point-of-care tests showed comparable performance, with sensitivities and specificities in the 60%-70% range when used alone and increasing to 80% when used in combination. The new point-of-care tests may serve a role at the bedside in those with clinical suspicion of IPA.

Cysteine proteases in protozoan parasites.

Cysteine proteases (CPs) play key roles in the pathogenesis of protozoan parasites, including cell/tissue penetration, hydrolysis of host or parasite proteins, autophagy, and evasion or modulation of the host immune response, making them attractive chemotherapeutic and vaccine targets. This review highlights current knowledge on clan CA cysteine proteases, the best-characterized group of cysteine proteases, from 7 protozoan organisms causing human diseases with significant impact: Entamoeba histolytica, Leishmania species (sp.), Trypanosoma brucei, T. cruzi, Cryptosporidium sp., Plasmodium sp., and Toxoplasma gondii. Clan CA proteases from three organisms (T. brucei, T. cruzi, and Plasmodium sp.) are well characterized as druggable targets based on in vitro and in vivo models. A number of candidate inhibitors are under development. CPs from these organisms and from other protozoan parasites should be further characterized to improve our understanding of their biological functions and identify novel targets for chemotherapy.

Automated Real-Time Collection of Pathogen-Specific Diagnostic Data: Syndromic Infectious Disease Epidemiology.

BACKGROUND: Health care and public health professionals rely on accurate, real-time monitoring of infectious diseases for outbreak preparedness and response. Early detection of outbreaks is improved by systems that are comprehensive and specific with respect to the pathogen but are rapid in reporting the data. It has proven difficult to implement these requirements on a large scale while maintaining patient privacy. OBJECTIVE: The aim of this study was to demonstrate the automated export, aggregation, and analysis of infectious disease diagnostic test results from clinical laboratories across the United States in a manner that protects patient confidentiality. We hypothesized that such a system could aid in monitoring the seasonal occurrence of respiratory pathogens and may have advantages with regard to scope and ease of reporting compared with existing surveillance systems. METHODS: We describe a system, BioFire Syndromic Trends, for rapid disease reporting that is syndrome-based but pathogen-specific. Deidentified patient test results from the BioFire FilmArray multiplex molecular diagnostic system are sent directly to a cloud database. Summaries of these data are displayed in near real time on the Syndromic Trends public website. We studied this dataset for the prevalence, seasonality, and coinfections of the 20 respiratory pathogens detected in over 362,000 patient samples acquired as a standard-of-care testing over the last 4 years from 20 clinical laboratories in the United States. RESULTS: The majority of pathogens show influenza-like seasonality, rhinovirus has fall and spring peaks, and adenovirus and the bacterial pathogens show constant detection over the year. The dataset can also be considered in an ecological framework; the viruses and bacteria detected by this test are parasites of a host (the human patient). Interestingly, the rate of pathogen codetections, on average 7.94% (28,741/362,101), matches predictions based on the relative abundance of organisms present. CONCLUSIONS: Syndromic Trends preserves patient privacy by removing or obfuscating patient identifiers while still collecting much useful information about the bacterial and viral pathogens that they harbor. Test results are uploaded to the database within a few hours of completion compared with delays of up to 10 days for other diagnostic-based reporting systems. This work shows that the barriers to establishing epidemiology systems are no longer scientific and technical but rather administrative, involving questions of patient privacy and data ownership. We have demonstrated here that these barriers can be overcome. This first look at the resulting data stream suggests that Syndromic Trends will be able to provide high-resolution analysis of circulating respiratory pathogens and may aid in the detection of new outbreaks.

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection.

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

Evaluation of opt-out inpatient HIV screening at an urban teaching hospital.

This study evaluated opt-out inpatient HIV screening delivered by admitting physicians, and compared number of HIV tests and diagnoses to signs and symptoms-directed HIV testing (based on physician orders) in the emergency department (ED). The opt-out inpatient HIV screening program was conducted over a one year period in patients who were admitted to the 386-bed University of California San Diego (UCSD) teaching hospital. Numbers of HIV tests and diagnoses were compared to those observed among ED patients who underwent physician-directed HIV testing during the same time period. Survey data were collected from a convenience sample of patients and providers regarding the opt-out testing program. Among 8488 eligible inpatients, opt-out HIV testing was offered to 3017 (36%) patients, and rapid antibody testing was performed in 1389 (16.4%) inpatients, resulting in 6 (0.4% of all tests) newly identified HIV infections (5/6 were admitted through the ED). Among 27,893 ED patients, rapid antibody testing was performed in 88 (0.3%), with 7 (8.0% of all tests) new HIV infections identified. HIV diagnoses in the ED were more likely to be men who have sex with men (MSM) (p = 0.029) and tended to have AIDS-related opportunistic infections (p = 0.103) when compared to HIV diagnoses among inpatients. While 85% of the 150 physicians who completed the survey were aware of the HIV opt-out screening program, 44% of physicians felt that they did not have adequate time to consent patients for the program, and only 30% agreed that a physician is best-suited to consent patients. In conclusion, the yield of opt-out HIV rapid antibody screening in inpatients was comparable to the national HIV prevalence average. However, uptake of screening was markedly limited in this setting where opt-out screening was delivered by physicians during routine care, with limited time resources being the major barrier.

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.

Synthesis and Evaluation of Oxyguanidine Analogues of the Cysteine Protease Inhibitor WRR-483 against Cruzain.

A series of oxyguanidine analogues of the cysteine protease inhibitor WRR-483 were synthesized and evaluated against cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi. Kinetic analyses of these analogues indicated that they have comparable potency to previously prepared vinyl sulfone cruzain inhibitors. Co-crystal structures of the oxyguanidine analogues WRR-666 (4) and WRR-669 (7) bound to cruzain demonstrated different binding interactions with the cysteine protease, depending on the aryl moiety of the P1' inhibitor subunit. Specifically, these data demonstrate that WRR-669 is bound noncovalently in the crystal structure. This represents a rare example of noncovalent inhibition of a cysteine protease by a vinyl sulfone inhibitor.

Immunostaining Detection of Cytomegalovirus in Gastrointestinal Biopsies: Clinicopathological Correlation at a Large Academic Health System.

BACKGROUND: Cytomegalovirus (CMV) infection can be asymptomatic in healthy individuals but may cause serious complications in immunocompromised patients. We investigated the clinicopathological correlation of CMV in gastrointestinal (GI) biopsies at our institute between January 1, 2013 and December 31, 2015. METHODS: A total of 105 non-neoplastic GI biopsies tested positive for CMV by immunohistochemistry (IHC). The IHC results were stratified as "true positive" if > 2 cells stained, or "rare positive" if only 1 - 2 cells stained. Clinical information including comorbidities, serum CMV viral loads, and treatment was reviewed and correlated. RESULTS: Overall 1% of all GI biopsies were positive for CMV by immunostaining. The most frequently involved organ was colon, followed by esophagus, stomach, ileum and duodenum. When > 2 cells were stained positive, serum CMV viral loads were positive in 52.2%, negative in 17.2%, and not tested in 27.6% of cases. When only 1 - 2 cells stained positive, CMV viral loads were positive in 23.4%, negative in 25.5%, and not tested in 51.1% of cases. We further showed that clinical management of CMV differs based on both pathological findings and underlying diseases. CONCLUSIONS: The role of CMV in GI biopsies remains controversial. We propose an algorithm of performing CMV immunostaining based on clinicopathological correlation.

New drug target in protozoan parasites: the role of thioredoxin reductase.

Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin's anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E. histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E. histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

Hsp90 inhibitors as new leads to target parasitic diarrheal diseases.

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.

Reprofiled drug targets ancient protozoans: drug discovery for parasitic diarrheal diseases.

Recently, we developed a novel automated, high throughput screening (HTS) methodology for the anaerobic intestinal parasite Entamoeba histolytica. We validated this HTS platform by screening a chemical library containing US Food and Drug Administration (FDA)-approved drugs and bioactive compounds. We identified an FDA-approved drug, auranofin, as most active against E. histolytica both in vitro and in vivo. Our cell culture and animal studies indicated that thioredoxin reductase, an enzyme involved in reactive oxygen species detoxification, was the target for auranofin in E. histolytica. Here, we discuss the rationale for drug development for three parasites which are major causes of diarrhea worldwide, E. histolytica, Giardia lamblia and Cryptosporidium parvum and extend our current finding of antiparasitic activity of auranofin to Entamoeba cysts, G. lamblia and C. parvum. These studies support the use of HTS assays and reprofiling FDA-approved drugs for new therapy for neglected tropical diseases.

Human milk oligosaccharides reduce Entamoeba histolytica attachment and cytotoxicity in vitro.

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infant's intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasite Entamoeba histolytica, as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) and N-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site of E. histolytica infection. The objective of the present study was to investigate whether HMO reduce E. histolytica attachment and cytotoxicity. Our in vitro results show that physiological concentrations of isolated, pooled HMO detach E. histolytica by more than 80 %. In addition, HMO rescue E. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80 % of the HT-29 cells, while HMO with fucose α1-2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolished E. histolytica attachment and cytotoxicity at 8 mg/ml. Although our results need to be confirmed in vivo, they may provide one explanation for why breast-fed infants are at lower risk of E. histolytica infections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents.

Effect of vinyl sulfone inhibitors of cysteine proteinases on Tritrichomonas foetus infection.

Tritrichomonas foetus is a sexually transmitted protozoon that causes genital inflammation and adverse pregnancy outcomes in cattle. Cysteine proteinases (CPs) released by T. foetus degrade immunoglobulin G (IgG) antibodies, complement component 3 and matrix proteins as well as inducing apoptosis of bovine genital epithelial cells. In this study, the efficacies of the vinyl sulfone CP inhibitors K11777 and WRR-483 were tested against CPs of T. foetus. The activity of secreted T. foetus CPs in culture supernatants was decreased in the presence of vinyl sulfone inhibitors. Inhibitor K11777 reduced the in vitro cytopathogenic effects of T. foetus in bovine foetal trophoblast cells, which are relevant target cells since this pathogen interferes with pregnancy. Pre-treatment of T. foetus prior to intravaginal inoculation diminished genital infection in a murine model. Therefore, vinyl sulfone CP inhibitors reduce several effects of T. foetus-secreted CPs, including cytotoxicity on relevant target host cells and genital infection in a murine model. These inhibitors have potential as chemotherapeutic agents against bovine trichomoniasis. Generalisation to human trichomoniasis requires further study.

Entamoeba histolytica induces intestinal cathelicidins but is resistant to cathelicidin-mediated killing.

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.

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.

Cyanide produced by human isolates of Pseudomonas aeruginosa contributes to lethality in Drosophila melanogaster.

Some Pseudomonas aeruginosa strains are cyanogenic, and cyanide may contribute to the bacterium's virulence. Using human isolates of P. aeruginosa, we have shown that Drosophila melanogaster suspended above cyanogenic strains become motionless and develop bradycardia and that flies injected with cyanogenic bacterial strains die more rapidly than those injected with noncyanogenic strains. Flies exposed to cyanogenic strains had high cyanide and low adenosine triphosphate (ATP) concentrations in body extracts, and treatment with a cyanide antidote equalized survival of flies injected with cyanogenic and noncyanogenic strains. P. aeruginosa PAO1 strain with a mutation in the hydrogen cyanide synthase gene cluster was much less toxic to flies than the parental cyanogenic strain or 2 knock-in strains. Transgenic flies overexpressing rhodanese, which detoxifies cyanide by converting it to thiocyanate, were resistant to cyanide and the increased virulence of cyanogenic strains. We conclude that D. melanogaster is a good model for studying cyanide produced by P. aeruginosa.

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

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.

Cathepsin Cs are key for the intracellular survival of the protozoan parasite, Toxoplasma gondii.

Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli beta-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.