Complete development of Cryptosporidium parvum in rabbit chondrocytes (VELI cells)

Cryptosporidium parvum is an intracellular protozoan parasite that causes severe infection in humans and animals. The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. The aim of this study was to demonstrate that C. parvum can complete its entire life cycle-from sporozoite to infective oocyst-in VELI cells (a line derived from primary culture of rabbit auricular chondrocytes). Successful infections were produced by inoculating cell cultures. Infection of MDCK, HTC-8 and VELI cells with C. parvum closely paralleled in vivo infections with regard to host cell location and chronology of parasite development. Oocysts which were produced in VELI cells were infective for infant NMRI mice. The growth of C. parvum in VELI cells provides a model, both simple and inexpensive, for testing anticryptosporidial drugs and studying host-parasite interactions.     

Molecular targets for detection and immunotherapy in Cryptosporidium parvum

Cryptosporidium parvum is an obligate protozoan parasite responsible for the diarrheal illness cryptosporidiosis in humans and animals. Although C. parvum is particularly pathogenic in immunocompromised hosts, the molecular mechanisms by which C. parvum invades the host epithelial cells are not well understood. Characterization of molecular-based antigenic targets of C. parvum is required to improve the specificity of detection, viability assessments, and immunotherapy (treatment). A number of zoite surface (glyco)proteins are known to be expressed during, and believed to be involved in, invasion and infection of host epithelial cells. In the absence of protective treatments for this illness, antibodies targeted against these zoite surface (glyco)proteins offers a rational approach to therapy. Monoclonal, polyclonal and recombinant antibodies represent useful immunotherapeutic means of combating infection, especially when highly immunogenic C. parvum antigens are utilized as targets. Interruption of life cycle stages of this parasite via antibodies that target critical surface-exposed proteins can potentially decrease the severity of disease symptoms and subsequent re-infection of host tissues. In addition, development of vaccines to this parasite based on the same antigens may be a valuable means of preventing infection. This paper describes many of the zoite surface glycoproteins potentially involved in infection, as well as summarizes many of the immunotherapeutic studies completed to date. The identification and characterization of antibodies that bind to C. parvum-specific cell surface antigens of the oocyst and sporozoite will allow researchers to fully realize the potential of molecular-based immunotherapy to this parasite.     

Treatment with agmatine inhibits Cryptosporidium parvum infection in infant mice

Cryptosporidium parvum is an intracellular protozoan parasite that causes enteric infection and diarrhea in a wide range of mammalian hosts, including humans and economically important livestock species. There are no effective vaccines or drug treatments available for cryptosporidiosis. Cryptosporidium parvum utilizes a unique metabolic pathway for the synthesis of polyamines, forming agmatine as an intermediary metabolite. We treated infant mice with oral doses of agmatine for 2 days before, the day of, and 5 days following experimental infection with C. parvum. Mice treated with agmatine were significantly less infected with C. parvum than were control mice receiving phosphate-buffered saline. Mice treated with agmatine only on the day of experimental infection with C. parvum were also significantly less infected than were control mice. These data suggest that exogenous agmatine alters the metabolism of C. parvum sufficient to interfere with its ability to colonize the mammalian intestine.     

Cryptosporidium parvum: the many secrets of a small genome

The coccidium Cryptosporidium parvum is an obligate intracellular parasite of the phylum Apicomplexa. It infects the gastrointestinal tract of humans and livestock, and represents the third major cause of diarrhoeal disease worldwide. Scarcely considered for decades due to its apparently non-pathogenic nature, C. parvum has been studied very actively over the last 15 years, after its medical relevance as a dangerous opportunistic parasite and widespread water contaminant was fully recognised. Despite the lack of an efficient in vitro culture system and appropriate animal models, significant advances have been made in this relatively short period of time towards understanding C. parvum biology, immunology, genetics and epidemiology. Until recently, very little was known about the genome of C. parvum, with even basic issues, such as the number and size of chromosomes, being the object of a certain controversy. With the advent of pulsed field gradient electrophoresis and the introduction of molecular biology techniques, the overall structure and fine organisation of the genome of C. parvum have started to be disclosed. Organised into eight chromosomes distributed in a very narrow range of molecular masses, the genome of C. parvum is one of the smallest so far described among unicellular eukaryotic organisms. Although fewer than 30 C. parvum genes have been cloned so far, information about the overall structure of the parasite genome has increased exponentially over the last 2 years. From the first karyotypic analyses to the recent development of physical maps for individual chromosomes, this review will try to describe the state-of-the-art of our knowledge on the nuclear genome of C. parvum and will discuss the available experimental evidence concerning the presence of extra-chromosomal elements.     

Oral dosing of neonatal mice with sucrose reduces infection with Cryptosporidium parvum

Cryptosporidium parvum is a significant cause of diarrheal disease in humans and economically important livestock species. There is no effective treatment available for this protozoan parasite. Mechanisms of intestinal colonization by C. parvum are not well understood, but it has been suggested that the parasite may utilize a lectin-like receptor. We used an infant mouse model to test whether high sugar concentrations in the intestine would affect in vivo colonization with C. parvum. We found that a single oral dose of sucrose, administered to mice at the time of, or 24 hr before, challenge with C. parvum significantly reduced infection. Significant reduction of infection was also seen in mice given isomaltose. Histologic examination of intestinal sections of mice treated with sucrose or isomaltose, but not other sugars, showed marked vacuolation of the small intestinal epithelium 1 day after treatment. Three days after treatment, tissue appeared normal. Thus, sucrose and, to a lesser extent, isomaltose reduced in vivo colonization with C. parvum and altered epithelial cell morphology in intestines of mice.     

Electron microscopic research on cryptosporidia. III. Parasite-host relations

A study was made of the host-parasite relationship with Cryptosporidium parvum (Apicomplexa, Sporozoa), which parasitizes the intestine of newborn rats experimentally infected with oocysts isolated from C. parvum-infected calves. The endogenous development of the parasite occurs extracytoplasmically in the microvillar compartment of the enterocytes. The formation of the extracytoplasmic parasitophorous vacuole (PV), like that surrounding the endogenous stages of C. parvum, is regarded as one of the possible and evolutionary established ways for the intracellular parasite to escape from the host cell lysosomal digestion. Special attention is paid to the attachment zone of C. parvum, where a multimembranous organelle is formed serving eventually as a feeder organelle. No other specialized cytostome, similar to the micropore of other coccidia, has been so far revealed in the growing stages of Cryptosporidium. The characteristic ultrastructural organization of the endogenous stages of C. parvum and of other Cryptosporidium species so far investigated, along with the peculiar structure of the cryptosporidia-surrounding PV, to say nothing of some other distinctive features--all this makes it possible to distinguish between the genus Cryptosporidium and other coccidian genera, and warrants the separation of the former into a separate family Cryptosporidiidae Léger, 1911. Unlike, the addition to this family, besides Cryptosporidium Tyzzer, 1910, of another genus, Epieimeria Dykova, 1981, on the ground of the "epicellular" localization of both the genera claimed by Levine (1984), seems hardly correct, due to the totally different patterns of ultrastructural organization and host-parasite relationship recently reported for Epieimeria anguillae by Molnar and Baska (1986).     

CCR5 is involved in controlling the early stage of Cryptosporidium parvum infection in neonates but is dispensable for parasite elimination

Chemokines play a critical role in immune cell trafficking and the transition from an innate to an acquired immune response. We analyzed host response in neonatal mice deficient in chemokine receptor CCR5 following infection with the intracellular protozoan parasite Cryptosporidium parvum. CCR5 neonatal mice had a higher parasite burden at the early stage of infection but eliminated the parasite as efficiently as their wild-type counterparts. The higher sensitivity of neonates at the beginning of infection was not due to an altered IFNgamma response. An increased CCR2-attracting chemokine response associated with the recruitment of CCR2-positive cells in the infected mucosa may have compensated for the absence of CCR5. A lack of CCR5 thus has an impact in the early stage of C. parvum infection in neonates, but this receptor is dispensable for subsequent parasite elimination.     

Caspase-dependent apoptosis during infection with Cryptosporidium parvum

The protozoan parasite Cryptosporidium parvum causes persistent diarrhea and malnutrition in children and the diarrhea-wasting syndrome in AIDS. No therapy exists for eliminating the parasite in the absence of a healthy immune response. Although it had been reported that infection of intestinal cell lines with C. parvum leads to host cell death, the mechanisms of cytolysis have not been characterized. We show here that infection with C. parvum leads to typical apoptotic nuclear condensation and DNA fragmentation in host cells. Both nuclear condensation and DNA fragmentation are inhibited by a caspase inhibitor, showing that caspases are involved in this type of apoptosis. Finally, blocking apoptosis with the caspase inhibitor increases the percentage of infected cells, suggesting that parasites may use apoptosis to exit from the infected cell or that the infected cells may eliminate the parasite through apoptosis. These results suggest that apoptosis could be involved in the pathogenesis of C. parvum infections in vivo, and raise the possibility that therapeutic interference with host cell death could alter the course of the pathology in vivo.     

Electron microscopic observation of the invasion process of Cryptosporidium parvum in severe combined immunodeficiency mice

Cryptosporidium parvum mainly invades the intestinal epithelium and causes watery diarrhea in humans and calves. However, the invasion process has not yet been clarified. In the present study, the invasion process of C. parvum in severe combined immunodeficiency (SCID) mice was examined. Infected mice were necropsied; the ilea were double-fixed routinely and observed by scanning and transmission electron microscopy. In addition, the microvillus membrane was observed by ruthenium red staining. Scanning electron micrographs showed elongation of the microvilli at the periphery of the parasite. The microvilli were shown to be along the surface of the parasite in higher magnification. Transmission electron microscopy confirmed that the invading parasites were located among microvilli. Parasites existed in the parasitophorous vacuole formed by the microvillus membrane. The parasite pellicle attached to the host cell membrane at the bottom of the parasite, and then the pellicle and host cell membrane became unclear. Subsequently, the pellicle became complicated and formed a feeder organelle. In addition, invasion of the parasite was not observed in either a microvillus or the cytoplasm of the host cell. Therefore, C. parvum invades among microvilli, is covered with membranes derived from numerous microvilli, and develops within the host cell.     

Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains

Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host-cell surface membranes including Gal/GalNAc-associated glycoproteins. Subsequent cellular entry of this parasite depends on host-cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3-kinase (PI-3K)/Cdc42-associated actin remodelling. How C. parvum hijacks these host-cell processes to facilitate its infection of target epithelia is unclear. Using specific probes to known components of sphingolipid-enriched membrane microdomains (SEMs), we detected aggregation of host-cell SEM components at infection sites during C. parvum infection of cultured human biliary epithelial cells (i.e. cholangiocytes). Activation and membrane translocation of acid-sphingomyelinase (ASM), an enzyme involved in SEM membrane aggregation, were also observed in infected cells. Pharmacological disruption of SEMs and knockdown of ASM via a specific small interfering RNA (siRNA) significantly decreased C. parvum attachment (by approximately 84%) and cellular invasion (by approximately 88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum-induced accumulation of Gal/GalNAc-associated glycoproteins at infection sites by approximately 90%. Disruption of SEMs and knockdown of ASM also significantly blocked C. parvum-induced activation of host-cell PI-3K and subsequent accumulation of Cdc42 and actin by up to 75%. Our results suggest an important role of SEMs for C. parvum attachment to and entry of host cells, likely via clustering of membrane-binding molecules and facilitating of C. parvum-induced actin remodelling at infection sites through activation of the PI-3K/Cdc42 signalling pathway.     

Electron tomographic and ultrastructural analysis of the Cryptosporidium parvum relict mitochondrion, its associated membranes, and organelles

Sporozoites of the apicomplexan Cryptosporidium parvum possess a small, membranous organelle sandwiched between the nucleus and crystalloid body. Based upon immunolabelling data, this organelle was identified as a relict mitochondrion. Transmission electron microscopy and tomographic reconstruction reveal the complex arrangement of membranes in the vicinity of this organelle, as well as its internal organization. The mitochondrion is enveloped by multiple segments of rough endoplasmic reticulum that extend from the outer nuclear envelope. In tomographic reconstructions of the mitochondrion, there is either a single, highly-folded inner membrane or multiple internal subcompartments (which might merge outside the reconstructed volume). The infoldings of the inner membrane lack the tubular "crista junctions" found in typical metazoan, fungal, and protist mitochondria. The absence of this highly conserved structural feature is congruent with the loss, through reductive evolution, of the normal oxidative phosphorylation machinery in C. parvum. It is proposed that the retention of a relict mitochondrion in C. parvum is a strategy for compartmentalizing away from the cytosol toxic ferrous iron and sulfide, which are needed for iron sulfur cluster biosynthesis, an essential function of mitochondria in all eukaryotes.     

Cryptosporidium parvum IMP dehydrogenase: identification of functional, structural, and dynamic properties that can be exploited for drug design

The protozoan parasite Cryptosporidium parvum causes severe enteritis with substantial morbidity and mortality among AIDS patients and young children. No fully effective treatment is available. C. parvum relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to produce guanine nucleotides and is highly susceptible to IMPDH inhibition. Furthermore, C. parvum obtained its IMPDH gene by lateral transfer from an epsilon-proteobacterium, suggesting that the parasite enzyme might have very different characteristics than the human counterpart. Here we describe the expression of recombinant C. parvum IMPDH in an Escherichia coli strain lacking the bacterial homolog. Expression of the parasite gene restores growth of this mutant on minimal medium, confirming that the protein has IMPDH activity. The recombinant protein was purified to homogeneity and used to probe the enzyme's mechanism, structure, and inhibition profile in a series of kinetic experiments. The mechanism of the C. parvum enzyme involves the random addition of substrates and ordered release of products with rate-limiting hydrolysis of a covalent enzyme intermediate. The pronounced resistance of C. parvum IMPDH to mycophenolic acid inhibition is in strong agreement with its bacterial origin. The values of Km for NAD and Ki for mycophenolic acid as well as the synergistic interaction between tiazofurin and ADP differ significantly from those of the human enzymes. These data suggest that the structure and dynamic properties of the NAD binding site of C. parvum IMPDH can be exploited to develop parasite-specific inhibitors.     

Do different parasite species interact in their effects on host fitness? A case study on parasites of the amphipod Paracalliope fluviatilis

There is a gap in our understanding of the relative and interactive effects of different parasite species on the same host population. Here we examine the effects of the acanthocephalan Acanthocephalus galaxii, an unidentified cyclophyllidean cestode, and the trematodes Coitocaecum parvum and Microphallus sp. on several fitness components of the amphipod Paracalliope fluviatilis, using a combination of infection surveys and both survival and behavioural trials. In addition to significant relationships between specific parasites and measures of amphipod survival, maturity, mating success and behaviour, interactions between parasite species with respect to amphipod photophilia were also significant. While infection by either A. galaxii or C. parvum was associated with increased photophilia, such increases were negated by co-infection with Microphallus sp. We hypothesize that this is due to the more subtle manipulative effect of A. galaxii and C. parvum being impaired by Microphallus sp. We conclude that the low frequency at which such double infections occur in our sampled population means that such interactions are unlikely to be important beyond the scale of the host individual. Whether or not this is generally true, implying that parasitological models and theory based on single parasite species studies do generally hold, requires cross-species meta-analytical studies.     

Characterization of a Cryptosporidium parvum Gene Encoding a Protein with Homology to Long Chain Fatty Acid Synthetase

ABSTRACT: We describe here the cloning, sequencing, and characterization of a novel Cryptosporidium parvum gene, encoding a protein with significant homology to the long-chain fatty acyl-CoA synthetase (LCFA, EC 6.2.13). The gene has an open reading frame of 2,301 bp, coding for a 766 amino acid polypeptide, and with an estimated MW of 86.1 kDa. By indirect immunofluorescence assay, monoclonal antibodies C3CE7 and ESD labeled the anterior pole of fixed C. parvum sporozoites and developmental stages in C. parvum-infected cultures at 24, 48, and 72 h post-infection. These monoclonal antibodies inhibited more than 3.5% of parasite growth in vitro. The effect of triacsin C, a potent selective inhibitor of LCFA synthetase, on parasite growth was assessed in cell culture; complete inhibition of parasite growth at 2.5 ug/inl was obtained with little evidence of drug-associated cytotoxicity. These results suggest that the fatty acyl-CoA synthetase may be a useful target in the development of selective inhibitors and immunologic interventions against C. parvum     

Current progress in the fatty acid metabolism in Cryptosporidium parvum

Cryptosporidium parvum is one of the apicomplexans that can cause severe diarrhea in humans and animals. The slow development of anti-cryptosporidiosis chemotherapy is primarily due to the poor understanding on the basic metabolic pathways in this parasite. Many well-defined or promising drug targets found in other apicomplexans are either absent or highly divergent in C. parvum. The recently discovered apicoplast and its associated Type II fatty acid synthetic enzymes in Plasmodium, Toxoplasma, and Eimeria apicomplexans are absent in C. parvum, suggesting this parasite is unable to synthesize fatty acids de novo. However, C. parvum possesses a giant Type I fatty acid synthase (CpFAS1) that makes very long chain fatty acids using mediate or long chain fatty acids as precursors. Cryptosporidium also contains a Type I polyketide synthase (CpPKS1) that is probably involved in the production of unknown polyketide(s) from a fatty acid precursor. In addition to CpFAS1 and CpPKS1, a number of other enzymes involved in fatty acid metabolism have also been identified. These include a long chain fatty acyl elongase (LCE), a cytosolic acetyl-CoA carboxylase (ACCase), three acyl-CoA synthases (ACS), and an unusual "long-type" acyl-CoA binding protein (ACBP), which allows us to hypothetically reconstruct the highly streamlined fatty acid metabolism in this parasite. However, C. parvum lacks enzymes for the oxidation of fatty acids, indicating that fatty acids are not an energy source for this parasite. Since fatty acids are essential components of all biomembranes, molecular and functional studies on these critical enzymes would not only deepen our understanding on the basic metabolism in the parasites, but also point new directions for the drug discovery against C. parvum and other apicomplexan-based diseases.     

Detection of infectious Cryptosporidium parvum oocysts in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule)

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10(3) oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.     

Cryptosporidium parvum attachment to and internalization by human biliary epithelia in vitro: a morphologic study

To explore the mechanisms by which Cryptosporidium parvum infects epithelial cells, we performed a detailed morphological study by serial electron microscopy to assess attachment to and internalization of biliary epithelial cells by C. parvum in an in vitro model of human biliary cryptosporidiosis. When C. parvum sporozoites initially attach to the host cell membrane, the rhoptry of the sporozoite extends to the attachment site; both micronemes and dense granules are recruited to the apical complex region of the attached parasite. During internalization, numerous vacuoles covered by the parasite's plasma membrane are formed and cluster together to establish a preparasitophorous vacuole. This preparasitophorous vacuole comes in contact with host cell membrane to form a host cell-parasite membrane interface, beneath which an electron-dense band begins to appear within the host cell cytoplasm. Simultaneously, host cells display membrane protrusion along the edge of the host cell-parasite membrane interface, resulting in the formation of a mature parasitophorous vacuole that completely covers the parasite. During internalization, vacuole-like structures appear in the apical complex region of the attached sporozoite, which bud out into host cells. A tunnel directly connecting the parasite to the host cell cytoplasm forms during internalization and remains when the parasite is totally internalized. Immunoelectron microscopy showed that sporozoite-associated proteins were localized along the dense band and at the parasitophorous vacuole membrane. These morphological observations provide evidence that secretion of parasite apical organelles and protrusion of host cell membrane play an important role in the attachment and internalization of host epithelial cells by C. parvum.