Saul Tzipori

The genome of Cryptosporidium hominis

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa—protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome ∼9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

Evidence for the shikimate pathway in apicomplexan parasites

Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently,. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals,. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

Cryptosporidiosis: biology, pathogenesis and disease.

Ninety-five years after discovery and after more than two decades of intense investigations, cryptosporidiosis, in many ways, remains enigmatic. Cryptosporidium infects all four classes of vertebrates and most likely all mammalian species. The speciation of the genus continues to be a challenge to taxonomists, compounded by many factors, including current technical difficulties and the apparent lack of host specificity by most, but not all, isolates and species.

Cryptosporidiosis in Perspective

Publisher Summary This chapter focuses on cryptosporidiosis in perspective. The significance of cryptosporidiosis in humans, however, depends on the severity of the disease it produces and the incidence in population, both of which are unknown. The frequency of cryptosporidiosis is highest in children aged between six months and three years. The chapter discusses these epidemiological observations and more accurate information regarding the morphology and the life-cycle of Cryptosporidium. Detailed studies on the life-cycle and the ultrastructure have provided a better understanding of the biology of the parasite and have highlighted its unique characteristics. Of these, the existence of sporulated, thin-walled oocysts, and their independence of reducing conditions for excystation are of major importance and may explain the occurrence of autoinfection and persistent infection. Cryptosporidium has numerous characteristics that set it apart from the rest of that subclass. The parasites ability to maintain persistent infection in certain individuals and its astonishingly stubborn resistance to chemotherapy are serious medical problems that need to be addressed in the future.

Natural History and Biology of Cryptosporidium parvum

The taxonomy of the genus Cryptosporidium remains ambiguous, because the current criteria for speciation are insufficient to validate the 6-8 named species. Cross-transmission experiments have shown varying and conflicting results, and the limited genetic data available do not necessarily support currently proposed species designations. The reasons for this ambiguity lie with the ubiquitous nature of Cryptosporidium, probably infecting all vertebrates and variety of tissues therein, and the absence of reference strains with defined virulence attributes that can be linked to genetic markers for comparative analysis. The inability to classify oocysts or confidently to identify their origin, implicate oocysts from all sources as hazardous to humans. Another major issue is the unusual degree of resistance that Cryptosporidium has shown to antiprotozoan and antimicrobial agents. The intracellular but extracytoplasmic domain the parasite occupies is in itself a significant barrier to drug entry. In support of this we outline how the intracellular niche of this parasite differs from the related Apicomplexans, Plasmodium and Toxoplasma, and delineate why the feeder organelle membrane, rather than, or in addition to, the parasitophorous membrane, is the major portal of nutrient entry for Cryptosporidium. The broad conclusion is that anticryptosporidial agents will have to enter the parasite via the multiple apical membranes that camouflage the parasite, or via the host cell, possibly transported by vesicles to the feeder organelle membrane. This may have major implications for rational drug discovery and design.

Prevalence of Enterocytozoon bieneusi in Swine: an 18-Month Survey at a Slaughterhouse in Massachusetts

ABSTRACT Slaughterhouse pig samples were analyzed by PCR for Enterocytozoon bieneusi infection. Thirty-two percent were found to be positive, with rates being higher over the summer months. Three isolates from pigs were identical in their ribosomal internal transcribed spacer sequence to human E. bieneusi type D, two were identical to type F (from a pig), and nine were previously unreported. The viability of these spores was demonstrated by their ability to infect gnotobiotic piglets. The presence of the infection in liver was shown by in situ hybridization.

Molecular Cloning and Expression of a Gene Encoding Cryptosporidium parvum Glycoproteins gp40 and gp15

ABSTRACT Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediateC. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein in C. parvum attachment to and invasion of host cells. We have cloned and sequenced a gene designated Cpgp40/15 that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated in C. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bp Cpgp40/15revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predicted O-glycosylation sites, a single potential N-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy ofCpgp40/15 in the C. parvum genome, and this gene does not contain introns. Our data indicate that the twoCpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis.

A hundred-year retrospective on cryptosporidiosis

Tyzzer discovered the genus Cryptosporidium a century ago, and for almost 70 years cryptosporidiosis was regarded as an infrequent and insignificant infection that occurred in the intestines of vertebrates and caused little or no disease. Its association with gastrointestinal illness in humans and animals was recognized only in the early 1980s. Over the next 25 years, information was generated on the diseases epidemiology, biology, cultivation, taxonomy and development of molecular tools. Milestones include: (i) recognition in 1980 of cryptosporidiosis as an acute enteric disease; (ii) its emergence as a chronic opportunistic infection that complicates AIDS; (iii) acknowledgement of impact on the water industry once it was shown to be waterborne; and (iv) study of Cryptosporidium genomics.

Treatment with bovine hyperimmune colostrum of cryptosporidial diarrhea in AIDS patients.

Cryptosporidium parvum may cause severe, debilitating diarrhea in patients with AIDS. Recent anecdotal reports have suggested that hyperimmune bovine colostrum may be effective. We conducted a double-blind, controlled pilot study of hyperimmune bovine colostrum for diarrhea due to cryptosporidiosis in five AIDS patients. The patients were randomized to receive either hyperimmune or control colostrum by continuous nasogastric infusion for 10 days. All stools were collected, graded, and weighed, and the concentration of oocysts excreted was determined daily. One of the three patients treated with hyperimmune colostrum had a reduction in diarrhea and in the concentration of oocysts excreted. A second treated patient had a modest decrease in the concentration of oocysts excreted. Two patients who received control colostrum also had decreases in the volume of diarrhea but no change in the concentration of oocysts excreted. We conclude that hyperimmune colostrum with high titers of specific anti-Cryptosporidium antibody could be effective in treating patients with cryptosporidiosis. However, more studies of cow colostral immunoglobulin need to be performed so that the efficacy of this treatment can be assessed more thoroughly.

Self-Regulation of Candida albicans Population Size during GI Colonization

Interactions between colonizing commensal microorganisms and their hosts play important roles in health and disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene, encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract. Counterintuitively, an efh1 null mutant exhibited increased colonization of the murine intestinal tract, a model of commensal colonization, whereas an EFH1 overexpressing strain exhibited reduced colonization of the intestinal tract and of the oral cavity of athymic mice, the latter situation modeling human mucosal candidiasis. When inoculated into the bloodstream of mice, both efh1 null and EFH1 overexpressing strains caused lethal infections. In contrast, other mutants are attenuated in virulence following intravenous inoculation but exhibited normal levels of intestinal colonization. Finally, although expression of several genes is dependent on transcription factor Efg1p during laboratory growth, Efg1p-independent expression of these genes was observed during growth within the murine intestinal tract. These results show that expression of EFH1 regulated the level of colonizing fungi, favoring commensalism as opposed to candidiasis. Also, different genes are required in different host niches and the pathway(s) that regulates gene expression during host colonization can differ from well-characterized pathways used during laboratory growth.