Microbial Health Risks of Cryptosporidium parvum and Giardia lamblia in Tropical Coastal Water in Araromi, Nigeria.

Objective: Giardia and Cryptosporidium are enteric protozoa that can cause a variety of gastrointestinal diseases, especially in vulnerable people like children, the elderly, and those with impaired immune systems. In order to ascertain the microbiological quality of the recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. This risk assessment is of great significance to human health protection against waterborne diseases. The aim of this study was to determine the microbial quality of recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. Methods: Microscopic examination of Cryptosporidium and Giardia oocysts were done. Results: Results revealed maximum occurrence of Cryptosporidium parvum (20 oocysts/100 mL) of water sample in the month of April and maximum occurrence of Giardia lamblia (300 cysts/100 mL) of water sample in the month of June. Additionally, according to Kolmogorov-Smirnov tests for normalcy Ho =0.05, Giardia lamblia and Cryptosporidium parvum were not regularly distributed in the water samples collected from the beach throughout the study period. The average likelihood of contracting Giardia lamblia and Cryptosporidium parvum infections after consuming 100 mL of beach water was 0.96 and 0.35, respectively. The risks of infection associated with Cryptosporidium parvum was lower than those associated with Giardia lamblia in water from the beach, but were both above the acceptable risk limit of 10-4. Conclusion: The results of this study indicate that Giardia and Cryptosporidium may represent serious health hazards to people who engage in aquatic activities. Adopting a comprehensive strategy that includes regular inspections, enhanced detection techniques, and the prevention of aquatic environment pollution may provide clean and safe recreational water for all, thereby safeguarding the public's health.

MiR-199a-3p regulates HCT-8 cell autophagy and apoptosis in response to Cryptosporidium parvum infection by targeting MTOR.

The microRNAs (miRNAs) of their hosts play an important role in regulating both the innate and adaptive immune responses to Cryptosporidium parvum infection. The mechanisms of autophagy and apoptosis are important components of the defense system against C. parvum infection. In this study, we investigate the role of miRNA-199a-3p in regulating MTOR-mediated autophagy and apoptosis in HCT-8 cells induced by C. parvum. The expression of miR-199a-3p increased at 3, 6 and 12 hours postinfection (hpi) but decreased at 24 and 48 hpi. The upregulation of miR-199a-3p promoted autophagy and apoptosis and limited the parasite burden in HCT-8 cells after C. parvum infection. The downregulation of miR-199a-3p inhibited the autophagy and apoptosis induced by C. parvum and enhanced the parasite burden in HCT-8 cells. A luciferase reporter showed that MTOR was a target gene of miR-199a-3p. Suppressed expression of MTOR by small interfering RNA (siRNA) promoted autophagy and apoptosis and limited C. parvum burden in HCT-8 cells. Co-transfection with miR-199a-3p inhibitor or si-mTOR revealed that miR-199a-3p regulates autophagy and apoptosis in HCT-8 cells through MTOR, to resist C. parvum infection. In conclusion, intestinal epithelial cells defend against C. parvum infection by regulating their autophagy and apoptosis through the miR-199a-3p-MTOR axis.

miR-181d targets BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to Cryptosporidium parvum infection via the intrinsic apoptosis pathway.

Cryptosporidium parvum is an important zoonotic pathogen that is studied worldwide. MicroRNAs (miRNAs) act as post-transcriptional regulators and may play a key role in modulating host epithelial responses following Cryptosporidium infection. Our previous study has shown that C. parvum downregulates the expression of miR-181d through the p50-dependent TLRs/NF-κB pathway. However, the mechanism by which miR-181d regulates host cells in response to C. parvum infection remains unclear. The present study found that miR-181d downregulation inhibited cell apoptosis and increased parasite burden in HCT-8 cells after C. parvum infection. Bioinformatics analysis and luciferase reporter assays have shown that BCL2 was a target gene for miR-181d. Moreover, BCL2 overexpression and miR-181d downregulation had similar results. To further investigate the mechanism by which miR-181d regulated HCT-8 cell apoptosis during C. parvum infection, the expression of molecules involved in the intrinsic apoptosis pathway was detected. Bax, caspase-9, and caspase-3 expression was decreased at 4, 8, 12, and 24 hpi and upregulated at 36 and 48 hpi. Interfering with the expression of miR-181d or BCL2 significantly affected the expression of molecules in the intrinsic apoptosis pathway. These data indicated that miR-181d targeted BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to C. parvum infection via the intrinsic apoptosis pathway. These results allowed us to further understand the regulatory mechanisms of host miRNAs during Cryptosporidium infection, and provided a theoretical foundation for the design and development of anti-cryptosporidiosis drugs.

Transcriptional control of the Cryptosporidium life cycle.

The parasite Cryptosporidium is a leading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

Cryptosporidium parvum disrupts intestinal epithelial barrier in neonatal mice through downregulation of cell junction molecules.

BACKGROUND: Cryptosporidium spp. cause watery diarrhea in humans and animals, especially in infants and neonates. They parasitize the apical surface of the epithelial cells in the intestinal lumen. However, the pathogenesis of Cryptosporidium-induced diarrhea is not fully understood yet. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we infected C57BL/6j neonatal mice with C. parvum IIa and IId subtypes, and examined oocyst burden, pathological changes, and intestinal epithelial permeability during the infection. In addition, transcriptomic analyses were used to study the mechanism of diarrhea induced by the C. parvum IId subtype. The neonatal mice were sensitive to both C. parvum IIa and IId infection, but the IId subtype caused a wide oocyst shedding window and maintained the high oocyst burden in the mice compared with the IIa subtype. In addition, the mice infected with C. parvum IId resulted in severe intestinal damage at the peak of infection, leading to increased permeability of the epithelial barrier. The KEGG, GO and GSEA analyses revealed that the downregulation of adherens junction and cell junction molecules at 11 dpi. Meanwhile, E-cadherin, which is associated with adherens junction, was reduced at the protein level in mouse ileum at peak and late infection. CONCLUSIONS/SIGNIFICANCE: C. parvum IId infection causes more severe pathological damage than C. parvum IIa infection in neonatal mice. Furthermore, the impairment of the epithelial barrier during C. parvum IId infection results from the downregulation of intestinal junction proteins.

Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection.

Introduction: Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored. Methods: Immune cells of the small intestinal Peyer's patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals. Results: Here, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection. Discussion: Considering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host-Cryptosporidium interactions in target species.

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.

Molecular Targets of the 5-Amido-Carboxamide Bumped Kinase Inhibitor BKI-1748 in Cryptosporidium parvum and HCT-8 Host Cells.

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.

Immunophenotyping of peripheral circulating lymphocytes and serum selenium levels in calves with neonatal diarrhea.

This work aims to: (1) elucidate the immune response exhibited by CD4 + and CD8 + T lymphocyte cells in response to various infectious agents in calves suffering with neonatal diarrhea; and (2) determine and investigate the association between serum selenium levels and T lymphocyte subtypes in neonatal calves afflicted with neonatal diarrhea and infected with various infectious agents. The study encompassed a cohort of 50 calves, encompassing both sexes and various breeds, within the neonatal age range (1-28 days old). Subdivided into distinct groups, the calves were categorized based on the causative agents of neonatal diarrhea, including Rotavirus (n = 10), Cryptosporidium parvum (C.parvum) (n = 10), Coronavirus (n = 5), Rotavirus+C.parvum (n = 5), and a Control group (n = 20). Blood samples were meticulously obtained from the vena jugularis of all animals utilizing specific techniques-8 ml in tubes devoid of anticoagulant and 3 ml in blood collection tubes containing EDTA. Serum selenium levels were analyzed by ICP-MS. Flow Cytometry device was used to determine CD4 + and CD8 +T lymphocyte levels. In this study, although there was no statistically significant difference in serum selenium levels between all study groups, it was found that the selenium level in the control group was not sufficient. CD4 T lymphocyte levels, the rotavirus+C.parvum group exhibited a statistically significant elevation compared to the coronavirus group. Regarding CD8 + T lymphocyte levels, the coronavirus group demonstrated a statistically significant increase when compared to the control group. In intragroup analyses of CD8 + T lymphocyte levels, the coronavirus group exhibited a significant elevation compared to the rotavirus group, C.parvum group, and the C.parvum + Rotavirus group. A significant negative correlation was detected between selenium levels and CD4 + T lymphocytes, while no correlation was found between CD8 + T lymphocytes. Fibrinogen concentration exhibited statistical significance, being higher in the Rotavirus group (p < 0.008) compared to the control group, in the C.parvum group (p < 0.004) compared to the control group, and in the Coronavirus group (p < 0.001) compared to the control group. The leukocyte count demonstrated statistical significance, being higher in the Rotavirus group compared to the control group (p < 0.001), in the Rotavirus+C.parvum group compared to the control group (p < 0.002), and in the Coronavirus group compared to the control group (p < 0.011). In conclusion, the data derived from this study illuminate discernible disparities in CD4 + and CD8 + T lymphocyte immune responses, contingent upon the specific etiological agent associated with neonatal diarrhea. Furthermore, the study underscores the importance of considering selenium deficiency as a relevant factor in calves affected by neonatal diarrhea.

Cryptosporidium parvum infection alters the intestinal mucosa transcriptome in neonatal calves: implications for immune function.

One of the leading causes of infectious diarrhea in newborn calves is the apicomplexan protozoan Cryptosporidium parvum (C. parvum). However, little is known about its immunopathogenesis. Using next generation sequencing, this study investigated the immune transcriptional response to C. parvum infection in neonatal calves. Neonatal male Holstein-Friesian calves were either orally infected (N = 5) or not (CTRL group, N = 5) with C. parvum oocysts (gp60 subtype IIaA15G2R1) at day 1 of life and slaughtered on day 7 after infection. Total RNA was extracted from the jejunal mucosa for short read. Differentially expressed genes (DEGs) between infected and CTRL groups were assessed using DESeq2 at a false discovery rate < 0.05. Infection did not affect plasma immunohematological parameters, including neutrophil, lymphocyte, monocyte, leucocyte, thrombocyte, and erythrocyte counts as well as hematocrit and hemoglobin concentration on day 7 post infection. The immune-related DEGs were selected according to the UniProt immune system process database and were used for gene ontology (GO) and pathway enrichment analysis using Cytoscape (v3.9.1). Based on GO analysis, DEGs annotated to mucosal immunity, recognizing and presenting antigens, chemotaxis of neutrophils, eosinophils, natural killer cells, B and T cells mediated by signaling pathways including toll like receptors, interleukins, tumor necrosis factor, T cell receptor, and NF-KB were upregulated, while markers of macrophages chemotaxis and cytosolic pattern recognition were downregulated. This study provides a holistic snapshot of immune-related pathways induced by C. parvum in calves, including novel and detailed feedback and feedforward regulatory mechanisms establishing the crosstalk between innate and adaptive immune response in neonate calves, which could be utilized further to develop new therapeutic strategies.

MiR-4521 affects the propagation of Cryptosporidium parvum in HCT-8 cells through targeting foxm1 by regulating cell apoptosis.

Cryptosporidium parvum could regulate the expression of microRNAs of epithelial cells to facilitate its intracellular propagation. MiR-4521 has been reported to play an important role during the development and progression of tumors and infectious diseases by regulating cell proliferation, apoptosis, and autophagy. However, the implication of miR-4521 during C. parvum infection was still unknown. In this study, the expression of miR-4521 was found to be upregulated in HCT-8 cells infected with C. parvum from 8 h post-infection (pi) to 48 hpi, and its upregulation would be related with the TLR/NF-κB signal pathway during C. parvum infection. One potential target of miR-4521, foxm1, was down-regulated in HCT-8 cells from 24 hpi to 48 hpi, and the expression of foxm1 was negatively regulated by miR-4521. The target relationship between miR-4521 and foxm1 was further validated by using dual luciferase reporter assay. Further studies showed that miR-4521 promoted the propagation of C. parvum in HCT-8 cells through targeting foxm1 by regulating BCL2-mediating cell apoptosis. These results contribute to further understanding of the regulatory mechanisms of host miRNAs during Cryptosporidium infection.

Case Report: The impact of severe cryptosporidiosis on the gut microbiota of a pediatric patient with CD40L immunodeficiency.

Cryptosporidium parvum is a protozoan parasite and one of the leading causes of gastroenteritis in the world, primarily affecting very young children and immunocompromised patients. While infection is usually self-limiting, it can become chronic and even lethal in these vulnerable populations, in whom Cryptosporidium treatments are generally ineffective, due to their acting in concert with a functioning immune system. Here, we describe a case of chronic cryptosporidiosis in a European child with severe CD40L immunodeficiency infected with Cryptosporidium parvum of the IIa20G1 subgenotype, a lineage which has thus far only ever been described in the Middle East. After years of on-off treatment with conventional and non-conventional anti-parasitic drugs failed to clear parasitosis, we performed targeted metagenomics to observe the bacterial composition of the patient's gut microbiota (GM), and to evaluate fecal microbiota transplantation (FMT) as a potential treatment option. We found that C. parvum infection led to significant shifts in GM bacterial composition in our patient, with consequent shifts in predicted intestinal functional signatures consistent with a state of persistent inflammation. This, combined with the patient's poor prognosis and increasing parasitic burden despite many rounds of anti-parasitic drug treatments, made the patient a potential candidate for an experimental FMT procedure. Unfortunately, given the many comorbidities that were precipitated by the patient's immunodeficiency and chronic C. parvum infection, FMT was postponed in favor of more urgently necessary liver and bone marrow transplants. Tragically, after the first liver transplant failed, the patient lost his life before undergoing FMT and a second liver transplant. With this case report, we present the first description of how cryptosporidiosis can shape the gut microbiota of a pediatric patient with severe immunodeficiency. Finally, we discuss how both our results and the current scientific literature suggest that GM modulations, either by probiotics or FMT, can become novel treatment options for chronic Cryptosporidium infection and its consequent complications, especially in those patients who do not respond to the currently available anti-parasitic therapies.

[Investigation of the Effect of Pasteurization on the Viability of Cryptosporidium parvum in Cow's Milk by Propidium Monoazide qPCR]. / Pastörizasyonun Inek Sütündeki Cryptosporidium parvum Canliligi Üzerine Etkisinin Propidium Monoazide qPCR ile Arastirilmasi.

Cow's milk, which is one of today's most important food sources, can be a reservoir for many pathogens that create a risk to public health. One of these pathogens is Cryptosporidium parvum. The oocysts of C.parvum, an obligate intracellular parasite, cause infection when ingested orally. The oocysts scattered around with the feces of infected cows or calves can contaminate raw milk and this is frequently seen in dairy farms. The aim of this study was to investigate the viability of C.parvum by propidium monoazide (PMA)-quantitative polymerase chain reaction (qPCR) method after heat treatment applied to contaminated raw cow's milk. For the study, 50 ml of unpasteurized cow's milk was contaminated with 5 X 105 C.parvum oocysts and portioned into 1.5 ml microcentrifuge tubes. Three groups, namely the control group, pasteurization group and boiling group were formed. No warming procedure was applied to the control group. In the pasteurization group, the milks in microcentrifuge tubes were poured into the wells of the dry block heater set to 71.7 °C and incubated for five seconds. At the end of the period, the milks were transferred to the wells of the cold metal tube, which was removed at -20 °C with the help of a micropipette, and incubated for five seconds. The milks in the boiling group were incubated for two minutes in a dry block heater set to 95 °C. After the heat treatment, the milks in microcentrifuge tubes were transferred to 10 ml centrifuge tubes, PBS was added to make the final volume 10 ml, and centrifuged at 4000 rpm for 20 minutes. After this process was repeated twice, 400 µl of PBS was added to the precipitate remaining at the bottom, and the precipitate was homogenized. One sample of each group was applied with PMA, while PMA was not applied to the other sample. PMA-applied samples were incubated for five minutes at room temperature and in the dark, and then exposed to UV light for five minutes in the device with cooling feature. The oocysts were collected by centrifugation at 5000 g for five minutes. After DNA isolation from oocysts, SYBR Green real time PCR (Rt-PCR) was performed using primers amplifying the COWP gene region. As a result of SYBR Green Rt-PCR, the mean Ct values of the control without PMA, pasteurization and boiling groups were determined as 25 ± 1.24, 23 ± 0.98 and 26 ± 1.03, respectively. While no peak was obtained in the boiling group after PMA application, the mean Ct values of the control and pasteurization groups were 28 ± 1.38 and 31 ± 1.46, respectively. As a result, it was concluded that live C.parvum cysts in milk could be detected by PMA-qPCR method and live oocysts could be found in pasteurized milk.

Mass Spectrometry Imaging of In Vitro Cryptosporidium parvum-Infected Cells and Host Tissue.

Cryptosporidium parvum is a zoonotic-relevant parasite belonging to the phylum Alveolata (subphylum Apicomplexa). One of the most zoonotic-relevant etiologies of cryptosporidiosis is the species C. parvum, infecting humans, cattle and wildlife. C. parvum-infected intestinal mucosa as well as host cells infected in vitro have not yet been the subject of extensive biochemical investigation. Efficient treatment options or vaccines against cryptosporidiosis are currently not available. Human cryptosporidiosis is currently known as a neglected poverty-related disease (PRD), being potentially fatal in young children or immunocompromised patients. In this study, we used a combination of atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) and liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine and locate molecular biomarkers in in vitro C. parvum-infected host cells as well as parasitized neonatal calf intestines. Sections of C. parvum-infected and non-infected host cell pellets and infected intestines were examined to determine potential biomarkers. Human ileocecal adenocarcinoma cells (HCT-8) were used as a suitable in vitro host cell system. More than a thousand different molecular signals were found in both positive- and negative-ion mode, which were significantly increased in C. parvum-infected material. A database search in combination with HPLC-MS/MS experiments was employed for the structural verification of markers. Our results demonstrate some overlap between the identified markers and data obtained from earlier studies on other apicomplexan parasites. Statistically relevant biomarkers were imaged in cell layers of C. parvum-infected and non-infected host cells with 5 µm pixel size and in bovine intestinal tissue with 10 µm pixel size. This allowed us to substantiate their relevance once again. Taken together, the present approach delivers novel metabolic insights on neglected cryptosporidiosis affecting mainly children in developing countries.

Effects of Cryptosporidium parvum infection on intestinal fungal microbiota in yaks (Bos grunniens).

During the last decade, researchers had started to focus on the relationship between intestinal parasitic infection and variation of intestinal microflora. Cryptosporidium is a widely known opportunistic and zoonotic pathogen. Several studies have shown that Cryptosporidium infection has impact to alter the gut microflora. However, there are only few studies referring to the fungal microflora changes in response to Cryptosporidium infection in highland ruminants. Therefore, the present study was performed for exploring the alternations of intestinal fungal microbiota in yaks after exposure to Cryptosporidium infection. In present study, Amplicon sequencing of ITS regions was used to study the variations of fungal microflora in yaks. After filtering the raw data, over 45 000 and 62 000 clean data were obtained in uninfected and infected yaks, respectively. By using alpha diversity analysis, it was found that there is no significant difference in the richness and evenness when positive samples were compared with negative ones, whereas intestinal fungal communities in different taxa in yaks were changed. The results of present study depicted that 2-phyla and 21-genera in the infected animals had significantly (P < 0.05) changed. These genera were Septoria, Coniothyrium, Cleistothelebolus, Bensingtonia, Cystobasidium, Filobasidium, Coprotus, Carex, Blumeria, Coprinellus, Leucosporidium, Phialophora, Isolepis, Ascobolus, Thecaphora, Mortierella, Urocystis, Symmetrospora and Lasiobolus. In addition, we found variations in 28 enzymes suggesting that the function of microbiota was also affected. It is concluded that there are drastic changes in the fungal microflora and microbiota functions after exposure to Cryptosporidium infection in yak. Our results help to focus on the prompt way for the development of new therapies to control Cryptosporidiosis.

MiR-3976 regulates HCT-8 cell apoptosis and parasite burden by targeting BCL2A1 in response to Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium is second only to rotavirus as a cause of moderate-to-severe diarrhea in young children. There are currently no fully effective drug treatments or vaccines for cryptosporidiosis. MicroRNAs (miRNAs) are involved in regulating the innate immune response to Cryptosporidium parvum infection. In this study, we investigated the role and mechanism of miR-3976 in regulating HCT-8 cell apoptosis induced by C. parvum infection. METHODS: Expression levels of miR-3976 and C. parvum burden were estimated using real-time quantitative polymerase chain reaction (RT-qPCR) and cell apoptosis was detected by flow cytometry. The interaction between miR-3976 and B-cell lymphoma 2-related protein A1 (BCL2A1) was studied by luciferase reporter assay, RT-qPCR, and western blotting. RESULTS: Expression levels of miR-3976 were decreased at 8 and 12 h post-infection (hpi) but increased at 24 and 48 hpi. Upregulation of miR-3976 promoted cell apoptosis and inhibited the parasite burden in HCT-8 cells after C. parvum infection. Luciferase reporter assay indicated that BCL2A1 was a target gene of miR-3976. Co-transfection with miR-3976 and a BCL2A1 overexpression vector revealed that miR-3976 targeted BCL2A1 and suppressed cell apoptosis and promoted the parasite burden in HCT-8 cells. CONCLUSIONS: The present data indicated that miR-3976 regulated cell apoptosis and parasite burden in HCT-8 cells by targeting BCL2A1 following C. parvum infection. Future study should determine the role of miR-3976 in hosts' anti-C. parvum immunity in vivo.

Cryptosporidium parvum hijacks a host's long noncoding RNA U90926 to evade intestinal epithelial cell-autonomous antiparasitic defense.

Cryptosporidium is a zoonotic apicomplexan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans. It is an important opportunistic pathogen in AIDS patients and a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. The intestinal epithelial cells provide the first line of defense against Cryptosporidium infection and play a central role in activating and regulating the host's antiparasitic response. Increasing evidence suggests that long noncoding RNAs (lncRNAs) participate in host-pathogen interactions and play a regulatory role in the pathogenesis of diseases but the underlying molecular mechanisms are not fully understood. We previously identified a panel of host lncRNAs that are upregulated in murine intestinal epithelial cells following Cryptosporidium infection, including U90926. We demonstrate here that U90926 is acting in a pro-parasitic manner in regulating intestinal epithelial cell-autonomous antiparasitic defense. Inhibition of U90926 resulted in a decreased infection burden of the parasite while overexpression of U90926 showed an increase in infection burden in cultured murine intestinal epithelial cells. Induction of U90926 suppressed transcription of epithelial defense genes involved in controlling Cryptosporidium infection through epigenetic mechanisms. Specifically, transcription of Aebp1, which encodes the Aebp1 protein, a potent modulator of inflammation and NF-κB signaling, was suppressed by U90926. Gain- or loss-of-function of Aebp1 in the host's epithelial cells caused reciprocal alterations in the infection burden of the parasite. Interestingly, Cryptosporidium carries the Cryptosporidium virus 1 (CSpV1), a double-stranded (ds) RNA virus coding two dsRNA fragments, CSpV1-dsRdRp and CSpV1-dsCA. Both CSpV1-dsRdRp and CSpV1-dsCA can be delivered into infected cells as previously reported. We found that cells transfected with in vitro transcribed CSpV1-dsCA or CSpV1-dsRdRp displayed an increased level of U90926, suggesting that CSpV1 is involved in the upregulation of U90926 during Cryptosporidium infection. Our study highlights a new strategy by Cryptosporidium to hijack a host lncRNA to suppress epithelial cell-autonomous antiparasitic defense and allow for a robust infection.

Circular RNA ciRS-7 affects the propagation of Cryptosporidium parvum in HCT-8 cells via regulating miR-135a-5p/stat1 axis.

Cryptosporidium spp. are protozoan parasites that mainly inhabit intestinal epithelial cells, causing diarrheal diseases in humans and a great number of animals. Cryptosporidium parvum is the most common zoonotic species, responsible for nearly 45% of human cryptosporidiosis worldwide. Understanding the interaction mechanisms between C. parvum and host gastrointestinal epithelial cells has significant implications to control cryptosporidiosis. One up-regulated circRNA ciRS-7 was found previously by our group to promote in vitro propagation of C. parvum in HCT-8 cells. In the present study, miR-135a-5p, was found to be a miRNA target of ciRS-7. Cryptosporidium parvum infection induced significantly down-regulation of miR-135a-5p and dramatic up-regulation of its potential target stat1 gene at mRNA and protein levels. Dual luciferase reporter assays validated the physical interactions between miR-135a-5p and stat1, and between ciRS-7 and miR-135a-5p. Further study revealed that ciRS-7 could sponge miR-135a-5p to positively regulate the protein levels of STAT1 and phosphorylated STAT1 (p-STAT1) and thus promote C. parvum propagation in HCT-8 cells. Our findings further reveal the mystery of regulatory roles of host circRNAs during Cryptosporidium infection, and provide a novel insight to develop strategies to control cryptosporidiosis.

Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis.

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

Structural Analyses of a Dominant Cryptosporidium parvum Epitope Presented by H-2Kb Offer New Options To Combat Cryptosporidiosis.

Cryptosporidium parvum has gained much attention as a major cause of diarrhea in the world, particularly in those with compromised immune systems. The data currently available on how the immune system recognizes C. parvum are growing rapidly, but we lack data on the interactions among host major histocompatibility complex (MHC) diversity and parasitic T-cell epitopes. To identify antigenic epitopes in a murine model, we performed systematic profiling of H-2Kb-restricted peptides by screening the dominant Cryptosporidium antigens. The results revealed that the glycoprotein-derived epitope Gp40/15-SVF9 induced an immunodominant response in C. parvum-recovered C57BL/6 mice, and injection of the cytotoxic-T-lymphocyte (CTL) peptide with the adjuvant activated peptide-specific CD8+ T cells. Notably, the SVF9 epitope was highly conserved across Cryptosporidium hominis, C. parvum, and many other Cryptosporidium species. SVF9 also formed stable peptide-MHC class I (MHC I) complexes with HLA-A*0201, suggesting cross-reactivity between H-2Kb and human MHC I specificities. Crystal structure analyses revealed that the interactions of peptide-MHC surface residues of H-2Kb and HLA-A*0201 are highly conserved. The hydrogen bonds of H-2Kb-SVF9 are similar to those of a dominant epitope presented by HLA-A*0201, which can be recognized by a public human T-cell receptor (TCR). Notably, we found double conformations in position 4 (P4), 5 (P5) of the SVF9 peptide, which showed high flexibility, and multiple peptide conformations generated more molecular surfaces that can potentially be recognized by TCRs. Our findings demonstrate that an immunodominant C. parvum epitope and its homologs from different Cryptosporidium species and subtypes can benefit vaccine development to combat cryptosporidiosis. IMPORTANCE Adaptive immune responses and T lymphocytes have been implicated as important mechanisms of parasite-induced protection. However, the role of CD8+ T lymphocytes in the resolution of C. parvum infection is largely unresolved. Our results revealed that the glycoprotein-derived epitope Gp40/15-SVF9 induced an immunodominant CD8+ T-cell response in C57BL/6 mice. Crystal structure analyses revealed that the interactions of the H-2Kb-SVF9 peptide are similar to those of a dominant epitope presented by HLA-A*0201, which can be recognized by human TCRs. In addition, we found double conformations of the SVF9 peptide, which showed high flexibility and multiple peptide conformations that can potentially be recognized by TCRs.