Functional characterization of CpADF, an actin depolymerizing factor protein in Cryptosporidium parvum.

Cryptosporidium is a highly pathogenic water and food-borne zoonotic parasitic protozoan that causes severe diarrhea in humans and animals. Apicomplexan parasites invade host cells via a unique motility process called gliding, which relies on the parasite's microfilaments. Actin depolymerizing factor (ADF) is a fibrous-actin (F-actin) and globular actin (G-actin) binding protein essential for regulating the turnover of microfilaments. However, the role of ADF in Cryptosporidium parvum (C. parvum) remains unknown. In this study, we preliminarily characterized the biological functions of ADF in C. parvum (CpADF). The CpADF was a 135-aa protein encoded by cgd5_2800 gene containing an ADF-H domain. The expression of cgd5_2800 gene peaked at 12 h post-infection, and the CpADF was located in the cytoplasm of oocysts, middle region of sporozoites, and cytoplasm of merozoites. Neutralization efficiency of anti-CpADF serum was approximately 41.30%. Actin sedimentation assay revealed that CpADF depolymerized but did not undergo cosedimentation with F-actin and its ability of F-actin depolymerization was pH independent. These results provide a basis for further investigation of the roles of CpADF in the invasion of C. parvum.

Eimeria spp. (Eimeriidae) in the migratory whooper swan (Cygnus cygnus) Linnaeus, 1758 (Anatidae) from Sanmenxia Swan Lake National Urban Wetland Park in the middle reaches of the Yellow River in China.

This study i dentifies four Eimeria spp. recorded from fecal samples of migratory whooper swans (Cygnus cygnus) in Sanmenxia Swan Lake National Urban Wetland Park in Sanmenxia city in the middle reaches of the Yellow River, China. Eimeria hermani, Eimeria nocens, Eimeria stigmosa, and Eimeria magnalabia were compatible in all characteristic features with their respective original descriptions. In addition to the preliminary morphological identification, this study provides a preliminary genotypic identification of these four Eimeria spp. via sequencing of the 18S rRNA, 28S rRNA, and COI gene loci that are suitable for the genotypic differentiation of these coccidia. This is the first report of molecular data for the four Eimeria spp. in migratory whooper swans. Finally, this study discusses the environmental risks of these coccidia for migratory whooper swans in Sanmenxia Swan Lake National Urban Wetland Park.

Identification of host protein ENO1 (alpha-enolase) interacting with Cryptosporidium parvum sporozoite surface protein, Cpgp40.

BACKGROUND: Cryptosporidium parvum is an apicomplexan zoonotic parasite causing the diarrheal illness cryptosporidiosis in humans and animals. To invade the host intestinal epithelial cells, parasitic proteins expressed on the surface of sporozoites interact with host cells to facilitate the formation of parasitophorous vacuole for the parasite to reside and develop. The gp40 of C. parvum, named Cpgp40 and located on the surface of sporozoites, was proven to participate in the process of host cell invasion. METHODS: We utilized the purified Cpgp40 as a bait to obtain host cell proteins interacting with Cpgp40 through the glutathione S-transferase (GST) pull-down method. In vitro analysis, through bimolecular fluorescence complementation assay (BiFC) and coimmunoprecipitation (Co-IP), confirmed the solid interaction between Cpgp40 and ENO1. In addition, by using protein mutation and parasite infection rate analysis, it was demonstrated that ENO1 plays an important role in the C. parvum invasion of HCT-8 cells. RESULTS: To illustrate the functional activity of Cpgp40 interacting with host cells, we identified the alpha-enolase protein (ENO1) from HCT-8 cells, which showed direct interaction with Cpgp40. The mRNA level of ENO1 gene was significantly decreased at 3 and 24 h after C. parvum infection. Antibodies and siRNA specific to ENO1 showed the ability to neutralize C. parvum infection in vitro, which indicated the participation of ENO1 during the parasite invasion of HCT-8 cells. In addition, we further demonstrated that ENO1 protein was involved in the regulation of cytoplasmic matrix of HCT-8 cells during C. parvum invasion. Functional study of the protein mutation illustrated that ENO1 was also required for the endogenous development of C. parvum. CONCLUSIONS: In this study, we utilized the purified Cpgp40 as a bait to obtain host cell proteins ENO1 interacting with Cpgp40. Functional studies illustrated that the host cell protein ENO1 was involved in the regulation of tight junction and adherent junction proteins during C. parvum invasion and was required for endogenous development of C. parvum.

Comparative proteomics reveals Cryptosporidium parvum infection disrupts cellular barriers.

Cryptosporidium is a protozoan parasite capable of infecting humans and animals and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms underlying invasive infection and its pathogenesis remain largely unknown. To better understand the molecular mechanism of the interaction between C. parvum and host cells, we profiled the changes of host cells membrane proteins extracted using native membrane protein extraction kit between C. parvum-infected HCT-8 cells and the control group after C. parvum infected 6 h combined with quantitative Tandem Mass Tags (TMT) liquid chromatography-dual mass spectrometry proteomic analysis. Among the 4844 quantifiable proteins identified, the expression levels of 625 were upregulated, and those of 116 were downregulated at 6 h post-infection compared with controls (1.5-fold difference in abundance, p < 0.05). Enrichment analysis of the function, protein domain and Kyoto Encyclopedia of Genes and Genomes pathway of the differentially expressed proteins revealed that the differentially expressed proteins were mainly related to biological functions related to the cytoskeleton and cytoplasmic matrix. We also found that infection with C. parvum may destroy HCT-8 intercellular space adhesion. Six proteins were further verified using quantitative real-time reverse transcription polymerase chain reaction and western blotting. Through systematic analysis of proteomics related to HCT-8 cell membranes infected by C. parvum, we found many host membrane proteins that can serve as potential receptors in C. parvum adhesion or invasion. C. parvum infection destroyed host cell barrier function and caused extensive changes in host cytoskeleton proteins, providing a deeper understanding of the molecules and their functions involved in the host-C. parvum interaction. SIGNIFICANCE: There is a lack of systematic research on the molecular mechanisms underlying the interaction of C. parvum with host cells. Changes of host cell membrane proteins after C. parvum infection may be used to examine the host cell receptors for parasite adhesion and invasion, and how the parasite interacts with these receptors. It is of great significance that host cells undergo membrane fusion to mediate invasion. Through proteomic studies on the host cell membrane after infection with HCT-8 cells by C. parvum, we observed disruption of the host cell cellular barrier function and widespread alteration of host cytoskeletal proteins caused by C. parvum infection, providing a deeper understanding of the molecules and their functions involved in host-C. parvum interaction.

Molecular characterization and zoonotic potential of Enterocytozoon bieneusi in ruminants in northwest China.

This study was conducted to examine the molecular characteristics and assess the zoonotic potential of Enterocytozoon bieneusi in ruminants in northwest China. A total of 1581 fresh fecal samples were collected from eight categories of ruminants. The E. bieneusi was screened and genotyped via nested polymerase chain reaction (PCR) amplification targeting the internal transcribed spacer (ITS) of the small subunit rRNA (ssu rRNA) gene. The result indicated that the average infection rate of E. bieneusi in ruminants was 16.0% (253/1581), with infection rates of E. bieneusi in Mongolian sheep, Mongolian goats, Chifeng cattle, red deer, alpine musk deer, and blue sheep at 21.8% (169/777), 8.2% (46/561), 25.9% (28/108), 13.2% (5/38), 20.0% (4/20), and 6.3% (1/16), respectively. The infections of E. bieneusi varied by different categories. For the different age groups, the infection rates in lambs (29.3%, 108/369) and calves (57.1%, 8/14) were significantly higher than that in ewes (21.1%, 215/1020) and cows (21.3%, 20/94). For the molecular characterization, diverse E. bieneusi ITS genotypes were identified, with a total of 13 genotypes were observed, including 10 known genotypes (BEB6, COS-I, J, CHC8, I, CHG1, BEB4, CHG3, CHS7, and NCF2) and 3 novel genotypes (CNR1 to CNR3). Genotype BEB6 was predominant (59.7%, 151/253). Phylogenetic analysis revealed that most E. bieneusi ITS genotypes clustered into group 2 and only one (NCF2) genotype belonged to group 1. The zoonotic genotypes identified in ruminants in the present study indicated the zoonotic potential of E. bieneusi. In addition, simultaneous identification of genotypes, such as BEB6, COS-I, and BEB4, in the same eco-geographical system indicated some host multiplicity transmission potential of E. bieneusi.