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The enteric pathogen Cryptosporidium parvum exports proteins into the cytosol of the infected host cell.

ABSTRACT: The parasite Cryptosporidium is responsible for diarrheal disease in young children causing death, malnutrition, and growth delay. Cryptosporidium invades enterocytes where it develops in a unique intracellular niche. Infected cells exhibit profound changes in morphology, physiology, and transcriptional activity. How the parasite effects these changes is poorly understood. We explored the localization of highly polymorphic proteins and found members of the Cryptosporidium parvum MEDLE protein family to be translocated into the cytosol of infected cells. All intracellular life stages engage in this export, which occurs after completion of invasion. Mutational studies defined an N-terminal host-targeting motif and demonstrated proteolytic processing at a specific leucine residue. Direct expression of MEDLE2 in mammalian cells triggered an ER stress response, which was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering parasite proteins into the infected enterocyte.

SUBMITTER: Dumaine JE

PROVIDER: S-EPMC8687662 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The diarrheal pathogen Cryptosporidium parvum exports proteins into the cytoplasm of the infected host cell

Project description:We found members of the polymorphic C. parvum MEDLE protein family to be targeted into the cytoplasm of infected cells. Direct expression of MEDLE2 in mammalian cells triggers an ER stress response that is also observed during infection.

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The diarrheal pathogen Cryptosporidium parvum exports proteins into the cytoplasm of the infected host cell

Project description:The diarrheal pathogen Cryptosporidium parvum exports proteins into the cytoplasm of the infected host cell

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Cryptosporidium parvum induces host cell actin accumulation at the host-parasite interface.

Project description:Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that Cryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein alpha-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including alpha-catenin, beta-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus, Cryptosporidium induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and alpha-actinin into a host-parasite junctional complex.

| S-EPMC97419 | biostudies-literature

Involvement of host cell integrin ?2 in Cryptosporidium parvum infection.

Project description:Cryptosporidium parvum is an opportunistic pathogen in AIDS patients. It is an intracellular but extracytoplasmic parasite residing in a host cell-derived parasitophorous vacuole. It is still poorly understood how this parasite interacts with host cells. We observed that expression of the integrin ?2 (ITGA2) gene in host cells was significantly upregulated upon C. parvum infection, and a higher level of ITGA2 protein was present in the parasite infection sites. The infection could be reduced by the treatment of antibodies against ITGA2 and integrin ?1 (ITGB1) subunits, as well as by type I collagen (an integrin ?2?1 ligand). We also generated stable knockdown of ITGA2 gene expression in HCT-8 cells and observed consistent reduction of parasite infection in these knockdown cells. Collectively, our evidence indicates that host cell ITGA2 might be involved in interacting with Cryptosporidium during infection, probably acting as part of the regulatory elements upstream of the reported recruiting and reorganization of F actin at the infection sites.

| S-EPMC3347445 | biostudies-literature

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

Project description:Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

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Cell sorting-assisted microarray profiling of host cell response to Cryptosporidium parvum infection.

Project description:To study the transcriptional response of mammalian cells to infection with the intracellular apicomplexan parasite Cryptosporidium parvum, infected and uninfected cells were recovered from C. parvum-infected cell monolayers. This approach, which contrasts with a more conventional experimental design that compares infected to uninfected cell monolayers, enabled the identification of functional categories of genes that are differentially transcribed as a direct consequence of the presence of intracellular parasites. Among several categories of upregulated genes, glycoprotein metabolism was significantly overrepresented. To investigate whether these transcriptional changes affected the composition of the surface of infected cells, cells were probed with fluorescently labeled lectins. Among a panel of seven lectins, soybean agglutinin, which recognizes N-acetyl-d-galactosamine, generated the largest difference in fluorescence between infected and uninfected cells. The origin of the fluorescent signal emitted by infected cells was further investigated and attributed to the overexpression of glycoprotein on the surface of infected cells, as well as the presence of glycoprotein located in the proximity of intracellular parasites.

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Transcriptome analysis of pig intestinal cell monolayers infected with Cryptosporidium parvum asexual stages.

Project description:BACKGROUND:Human cryptosporidiosis is caused primarily by two species of apicomplexan protozoa, Cryptosporidium parvum and C. hominis. In cultured cell monolayers, the parasite undergoes two generations of asexual multiplication (merogony). However, the proportion of parasites completing the life-cycle is low and insufficient to sustain continuous propagation. Due to the intracellular location of meronts and later life-cycle stages, oocyst and sporozoites are the only forms of the parasite that can readily be isolated. RESULTS:Research on the replicating forms of Cryptosporidium parasites and their interaction with the host cell remains challenging. Based on an RNA-Seq analysis of monolayers of pig epithelial cells infected with C. parvum, here we report on the impact of merogony on the host's gene regulation. Analysis of the transcriptome of infected and uninfected monolayers demonstrates a significant impact of the infection on host cell gene expression. A total of 813 genes were differentially expressed. Functional terms significantly altered in response to infection include phosphoprotein, RNA binding and acetylation. Upregulation of cell cycle pathways indicates an increase in mitosis. Notably absent from differentially enriched functional categories are stress- and apoptosis-related functions. The comparison of the combined host-parasite transcriptome reveals that C. parvum gene expression is less diverse than the host cell transcriptome and is highly enriched for genes encoding ribosomal functions, such as ribosomal proteins. CONCLUSIONS:These results indicate that C. parvum infection significantly changes host biological functions and provide new insight into gene functions driving early C. parvum intracellular development.

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Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells.

Project description:The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers the intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously.

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Characterization of 14-3-3 proteins from Cryptosporidium parvum.

Project description:UnlabelledThe parasite Cryptosporidium parvum has three 14-3-3 proteins: Cp14ε, Cp14a and Cp14b, with only Cp14ε similar to human 14-3-3 proteins in sequence, peptide-binding properties and structure. Structurally, Cp14a features the classical 14-3-3 dimer but with a uniquely wide pocket and a disoriented RRY triad potentially incapable of binding phosphopeptides. The Cp14b protein deviates from the norm significantly: (i) In one subunit, the phosphorylated C-terminal tail is bound in the binding groove like a phosphopeptide. This supports our binding study indicating this protein was stabilized by a peptide mimicking its last six residues. (ii) The other subunit has eight helices instead of nine, with αA and αB forming a single helix and occluding the peptide-binding cleft. (iii) The protein forms a degenerate dimer with the two binding grooves divided and facing opposite directions. These features conspire to block and disrupt the bicameral substrate-binding pocket, suggesting a possible tripartite auto-regulation mechanism that has not been observed previously.Enhanced versionThis article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

| S-EPMC3154893 | biostudies-literature

Evidence of host-associated populations of Cryptosporidium parvum in Italy.

Project description:Recent studies have revealed extensive genetic variation among isolates of Cryptosporidium parvum, an Apicomplexan parasite that causes gastroenteritis in both humans and animals worldwide. The parasite's population structure is influenced by the intensity of transmission, the host-parasite interaction, and husbandry practices. As a result, C. parvum populations can be panmictic, clonal, or even epidemic on both a local scale and a larger geographical scale. To extend the study of C. parvum populations to an unexplored region, 173 isolates of C. parvum collected in Italy from humans and livestock (calf, sheep, and goat) over a 10-year period were genotyped using a multilocus scheme based on 7 mini- and microsatellite loci. In agreement with other studies, extensive polymorphism was observed, with 102 distinct multilocus genotypes (MLGs) identified among 173 isolates. The presence of linkage disequilibrium, the confinement of MLGs to individual farms, and the relationship of many MLGs inferred using network analysis (eBURST) suggest a predominantly clonal population structure, but there is also evidence that part of the diversity can be explained by genetic exchange. MLGs from goats were found to differ from bovine and sheep MLGs, supporting the existence of C. parvum subpopulations. Finally, MLGs from isolates collected between 1997 and 1999 were also identified as a distinct subgroup in principal-component analysis and eBURST analysis, suggesting a continuous introduction of novel genotypes in the parasite population.

| S-EPMC3346357 | biostudies-literature

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