Prevalence and genotyping identification of Cryptosporidium in adult ruminants in central Iran.

BACKGROUND: Apicomplexan parasites of the genus Cryptosporidium infect a wide range of animal species as well as humans. Cryptosporidium spp. can cause life threatening diarrhea especially in young animals, children, immunocompromised patients and malnourished individuals. Asymptomatic cryptosporidial infections in animals can also occur, making these animals potential reservoirs of infection. METHODS: In the present study, a molecular survey of Cryptosporidium spp. in ruminants that were slaughtered for human consumption in Yazd Province, located in central Iran was conducted. Faeces were collected per-rectum from 484 animals including 192 cattle, 192 sheep and 100 goats. DNA was extracted from all samples and screened for Cryptosporidium by PCR amplification of the 18S rRNA gene. Positives were Sanger sequenced and further subtyped by sequence analysis of the 60 kDa glycoprotein (gp60) locus. RESULTS: In total, Cryptosporidium spp. were detected in 22 animals: C. andersoni and C. bovis in seven and two cattle faecal samples, respectively, C. ubiquitum in five sheep, and C. xiaoi in six sheep and two goat samples, respectively. To our knowledge, this study provides for the first time, molecular information concerning Cryptosporidium species infecting goats in Iran, and is also the first report of C. ubiquitum and C. xiaoi from ruminants in Iran. CONCLUSION: The presence of potentially zoonotic species of Cryptosporidium in ruminants in this region may suggest that livestock could potentially contribute to human cryptosporidiosis, in particular among farmers and slaughterhouse workers, in the area. Further molecular studies on local human populations are required to more accurately understand the epidemiology and transmission dynamics of Cryptosporidium spp. in this region.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Amelioration of Cryptosporidium parvum infection in vitro and in vivo by targeting parasite fatty acyl-coenzyme A synthetases.

BACKGROUND: Cryptosporidium is emerging as 1 of the 4 leading diarrheal pathogens in children in developing countries. Its infections in patients with AIDS can be fatal, whereas fully effective treatments are unavailable. The major goal of this study is to explore parasite fatty acyl-coenzyme A synthetase (ACS) as a novel drug target. METHODS: A colorimetric assay was developed to evaluate biochemical features and inhibitory kinetics of Cryptosporidium parvum ACSs using recombinant proteins. Anticryptosporidial efficacies of the ACS inhibitor triacsin C were evaluated both in vitro and in vivo. RESULTS: Cryptosporidium ACSs displayed substrate preference toward long-chain fatty acids. The activity of parasite ACSs could be specifically inhibited by triacsin C with the inhibition constant Ki in the nanomolar range. Triacsin C was highly effective against C. parvum growth in vitro (median inhibitory concentration, 136 nmol/L). Most importantly, triacsin C effectively reduced parasite oocyst production up to 88.1% with no apparent toxicity when administered to Cryptosporidium-infected interleukin 12 knockout mice at 8-15 mg/kg/d for 1 week. CONCLUSIONS: The findings of this study not only validated Cryptosporidium ACS (and related acyl-[acyl-carrier-protein]-ligases) as pharmacological targets but also indicate that triacsin C and analogues can be explored as potential new therapeutics against the virtually untreatable cryptosporidial infection in immunocompromised patients.

A novel calcium-dependent protein kinase inhibitor as a lead compound for treating cryptosporidiosis.

Cryptosporidium parasites infect intestinal cells, causing cryptosporidiosis. Despite its high morbidity and association with stunting in the developing world, current therapies for cryptosporidiosis have limited efficacy. Calcium-dependent protein kinases (CDPKs) are essential enzymes in the biology of protozoan parasites. CDPK1 was cloned from the genome of Cryptosporidium parvum, and potent and specific inhibitors have been developed based on structural studies. In this study, we evaluated the anti-Cryptosporidium activity of a novel CDPK1 inhibitor, 1294, and demonstrated that 1294 significantly reduces parasite infection in vitro, with a half maximal effective concentration of 100 nM. Pharmacokinetic studies revealed that 1294 is well absorbed, with a half-life supporting daily administration. Oral therapy with 1294 eliminated Cryptosporidium parasites from 6 of 7 infected severe combined immunodeficiency-beige mice, and the parasites did not recur in these immunosuppressed mice. Mice treated with 1294 had less epithelial damage, corresponding to less apoptosis. Thus, 1294 is an important lead for the development of drugs for treatment of cryptosporidiosis.

Identification and characterization of a novel calcium-activated apyrase from Cryptosporidium parasites and its potential role in pathogenesis.

Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.

Prodrug activation by Cryptosporidium thymidine kinase.

Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k(cat) = 0.28 s(-1) and K(m)(,ATP) = 140 microm are similar to those of human TK1, the value of K(m)(thymidine) = 48 microm is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.

Induction of arginase II by intestinal epithelium promotes the uptake of L-arginine from the lumen of Cryptosporidium parvum-infected porcine ileum.

OBJECTIVES: To determine the specific transport system activities and expression of transporter genes responsible for uptake of L-arginine from the lumen of normal and Cryptosporidium parvum-infected neonatal porcine ileum and the influence of L-arginine catabolic pathways on L-arginine uptake. METHODS: Intact sheets of ileal mucosa from control and C parvum-infected neonatal piglets were mounted in Ussing chambers and the uptake of 14C-L-arginine was determined under initial rate conditions and in the presence of transport system-selective inhibitors. Epithelial expression of L-arginine transporter genes was quantified by real-time reverse transcription polymerase chain reaction. L-Arginine catabolic enzyme expression was examined by immunoblotting epithelial lysates for arginase I and II. The role of intracellular catabolism in promoting the uptake of L-arginine was determined by pharmacological inhibition of nitric oxide synthase and arginase activities. RESULTS: C parvum-infected ileum transported L-arginine at rates equivalent to uninfected epithelium despite profound villous atrophy. This was attributed to enhanced uptake of L-arginine by individual epithelial cells in the infection. There were no differences in L-arginine transport system activities (y(+) and B(0, +)) or level of transporter gene expression (CAT-1, CAT-2A, and ATB(0, +)) between uninfected and C parvum-infected epithelial cells. However, infected epithelia had induced expression of the L-arginine hydrolytic enzyme arginase II and lower concentrations of L-arginine. Furthermore, transport of L-arginine by the infected epithelium was significantly inhibited by pharmacological blockade of arginase. CONCLUSIONS: Intracellular catabolism by arginase II, the induction of which has not been described previously for intestinal epithelium, facilitates uptake of L-arginine by infected epithelium using transport systems that do not differ from those of uninfected cells. Induction of arginase II may limit nitric oxide synthesis by competing with nitric oxide synthase for utilization of L-arginine or promote use of L-arginine for the synthesis of reparative polyamines.

Targeting a prokaryotic protein in a eukaryotic pathogen: identification of lead compounds against cryptosporidiosis.

Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. No vaccines exist against C. parvum, the drugs currently approved to treat cryptosporidiosis are ineffective, and drug discovery is challenging because the parasite cannot be maintained continuously in cell culture. Mining the sequence of the C. parvum genome has revealed that the only route to guanine nucleotides is via inosine-5'-monophosphate dehydrogenase (IMPDH). Moreover, phylogenetic analysis suggests that the IMPDH gene was obtained from bacteria by lateral gene transfer. Here we exploit the unexpected evolutionary divergence of parasite and host enzymes by designing a high-throughput screen to target the most diverged portion of the IMPDH active site. We have identified four parasite-selective IMPDH inhibitors that display antiparasitic activity with greater potency than paromomycin, the current gold standard for anticryptosporidial activity.

NF-kappaB-mediated expression of iNOS promotes epithelial defense against infection by Cryptosporidium parvum in neonatal piglets.

Cryptosporidium sp. parasitizes intestinal epithelium, resulting in enterocyte loss, villous atrophy, and malabsorptive diarrhea. We have shown that mucosal expression of inducible nitric oxide (NO) synthase (iNOS) is increased in infected piglets and that inhibition of iNOS in vitro has no short-term effect on barrier function. NO exerts inhibitory effects on a variety of pathogens; nevertheless, the specific sites of iNOS expression, pathways of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear. Using an in vivo model of Cryptosporidium parvum infection, we have examined the location, mechanism of induction, specificity, and consequence of iNOS expression in neonatal piglets. In acute C. parvum infection, iNOS expression predominated in the villous epithelium, was NF-kappaB dependent, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion but was not detrimental to maintenance of mucosal barrier function. Intensified parasitism could not be attributed to attenuated fluid loss or changes in epithelial proliferation or replacement rate, inasmuch as iNOS inhibition did not alter severity of diarrhea, piglet hydration, Cl- secretion, or kinetics of bromodeoxyuridine-labeled enterocytes. These findings suggest that induction of iNOS represents a nonspecific response of the epithelium that mediates enterocyte defense against C. parvum infection. iNOS did not contribute to the pathogenic sequelae of C. parvum infection.

Nitric oxide synthase stimulates prostaglandin synthesis and barrier function in C. parvum-infected porcine ileum.

Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PGE2, and increased mucosal permeability. Nonselective inhibition of NOS (NG-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PGE2. Selective inhibition of iNOS [L-N6-(1-iminoethyl)-L-lysine] accounted for approximately 50% of NOS-dependent PGE2 synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes.

Role of anti-oxidant preparation (oxi-guard) on Cryptosporidium parvum infection in albino mice.

Oxi-guard is a commercial anti-oxidant preparation in the form of capsules. One hundred and twenty albino mice were divided into three groups, first (50 mice) received oxi-guard for one week before infection with Cryptosporidium oocysts and continued for 50 days post-infection (P.I.), second (50 mice) did not receive oxi-guard and infected with Cryptosporidium oocysts and third (20 mice) was neither infected, nor receiving oxi-guard, a control group. Histopathological examination of small intestines and counting of endogenous stages of Cryptosporidium in the epithelial surface were done. Malonyldialdehyde (MDA) levels in small intestines were measured in all mice. It had been observed that the administration of oxi-guard led to significant increase in the numbers of endogenous stages of Cryptosporidium in the small intestines of group (1) in comparison with group (2). MDA levels showed a significant increase on the 15th, 20th and 25th days P.I. in group (2) and they did not show any significant changes in group (1) in comparison with the control group. As regards the activity of endogenous anti-oxidant enzymes in Cryptosporidium parvum oocysts, there was a significant increase in superoxide dismutase in group (2) of mice compared with its level in group (1). Finally, it can be concluded that the administration of oxi-guard had led to eradication of free radicals which in turn prevented their lethal effects on the parasite leading to increased parasitic colonization in the small intestine of mice.

[Cellular interactions in the intracellular parasitism of cryptosporidia. I. The effect of Cryptosporidium parvum on the phosphatase activity in the small intestine enterocytes of experimentally infected newborn rat pups]. / Kletochnye vzaimodeistviia pri vnutrikletochnom parazitirovanii kriptosporidii. I. Vliianie kriptosporidii Cryptosporidium parvum na fosfataznuiu aktivnost' v énterotsitakh tonkoi kishki éksperimental'no zarazhennykh novorozhdennykh krysiat.

Cytochemical methods for detection of non-specific phosphatases were employed at the light microscope level for identification of enzymatic activity in the small intestine of new-born rats (6--11 days old), both infected and non-infected with the intestinal coccidium Cryptosporidium parvum. In the new-born rats, the level of alkaline and especially acid phosphatase is originally very low, suggesting their insignificant involvement in digestion processes in suckling animals compared to rats of older age (3 month old). However, a heavy colonization of the brush border of the intestinal villi of the new-born rats with cryptosporidia results in obvious inactivation of phosphatases in the infected enterocytes, in contrast to the neighbouring parasite-free host cells. The general picture of metabolic interaction between cells of a unicellular parasite (C. parvum) and those of its metazoan host (rat) much resembles that observed in the course of Elmeria spp. infection, but differs from that induced by Toxoplasma gondii endogenous stages in the cat intestine. Details of cell interaction with intracellular parasitism need additional studies at the ultrastructural level.

Infection dynamics and clinical features of cryptosporidiosis in SCID mice.

Cryptosporidial infections in severe combined immune deficient (SCID) mice produce a chronic disease state which in the later stages leads to extraintestinal involvement and hepatic dysfunction. To further characterize the infection dynamics in this model and monitor the changes in the hepatic system, a dose titration of the oocyst inoculum was performed and alkaline phosphatase levels in the sera were assayed. Ten SCID mice per dose were inoculated with 10(3), 10(4), 10(5), 10(6), or 10(7) oocysts. Oocyst shedding in the feces was quantified by microscopic enumeration. Mice inoculated with 10(6) oocysts and those inoculated with 10(7) oocysts demonstrated similar oocyst shedding patterns, but the 10(7)-oocyst group exhibited signs of distress (e.g., weight loss and icterus) earlier. The intensity of the infection increased markedly approximately 14 days postinoculation (p.i.) and continued to increase steadily over the next 6 weeks. Inoculation with lower oocyst doses produced a delay in patency (e.g., it occurred 7 days later with the 10(5)-oocyst inoculum and 14 days later with the 10(4)-oocyst inoculum). Mean serum alkaline phosphatase levels in the 10(7)-oocyst group were more than twice control values at 5 weeks p.i. and continued to increase over the next 8 weeks. Oocyst doses and alkaline phosphatase levels were positively correlated with hepatobiliary colonization (r = 0.71) and liver necrosis (r = 0.65) at 13 weeks p.i. A strong positive correlation between hepatobiliary colonization and liver necrosis at 13 weeks p.i. (r = 0.87) was observed.