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.

Whole transcriptome analysis of HCT-8 cells infected by Cryptosporidium parvum.

BACKGROUND: Cryptosporidium species are zoonotic protozoans that are important causes of diarrhoeal disease in both humans and animals. Non-coding RNAs (ncRNAs) play an important role in the innate immune defense against Cryptosporidium infection, but the underlying molecular mechanisms in the interaction between human ileocecal adenocarcinoma (HCT-8) cells and Cryptosporidium species have not been entirely revealed. METHODS: The expression profiles of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) in the early phase of infection of HCT-8 cells with Cryptosporidium parvum and at 3 and 12 h post infection were analyzed using the RNA-sequencing technique. The biological functions of differentially expressed RNAs (dif-RNAs) were discovered through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The targeting relationships between three ncRNAs and mRNAs were analyzed using bioinformatics methods, followed by building a competing endogenous RNA (ceRNA) regulatory network centered on miRNAs. RESULTS: After strictly filtering the raw data, our analysis revealed 393 dif-lncRNAs, 69 dif-miRNAs and 115 dif-mRNAs at 3 hpi, and 450 dif-lncRNAs, 129 dif-miRNAs, 117 dif-mRNAs and one dif-circRNA at 12 hpi. Of these, 94 dif-lncRNAs, 24 dif-miRNAs and 22 dif-mRNAs were detected at both post-infection time points. Eleven dif-lncRNAs, seven dif-miRNAs, eight dif-mRNAs and one circRNA were randomly selected and confirmed using the quantitative real-time PCR. Bioinformatics analyses showed that the dif-mRNAs were significantly enriched in nutritional absorption, metabolic processes and metabolism-related pathways, while the dif-lncRNAs were mainly involved in the pathways related to the infection and pathogenicity of C. parvum (e.g. tight junction protein) and immune-related pathways (e.g. cell adhesion molecules). In contrast, dif-miRNAs and dif-circRNA were significantly enriched in apoptosis and apoptosis-related pathways. Among the downregulated RNAs, the miRNAs has-miR-324-3p and hsa-miR-3127-5p appear to be crucial miRNAs which could negatively regulate circRNA, lncRNA and mRNA. CONCLUSIONS: The whole transcriptome profiles of HCT-8 cells infected with C. parvum were obtained in this study. The results of the GO and KEGG pathway analyses suggest significant roles for these dif-RNAs during the course of C. parvum infection. A ceRNA regulation network containing miRNA at its center was constructed for the first time, with hsa-miR-324-3p and hsa-miR-3127-5p being the crucial miRNAs. These findings provide novel insights into the responses of human intestinal epithelial cells to C. parvum infection.

Putative SET-domain methyltransferases in Cryptosporidium parvum and histone methylation during infection.

Cryptosporidium parvum is a leading cause of diarrhoeal illness worldwide being a significant threat to young children and immunocompromised patients, but the pathogenesis caused by this parasite remains poorly understood. C. parvum was recently linked with oncogenesis. Notably, the mechanisms of gene expression regulation are unexplored in Cryptosporidium and little is known about how the parasite impact host genome regulation. Here, we investigated potential histone lysine methylation, a dynamic epigenetic modification, during the life cycle of the parasite. We identified SET-domain containing proteins, putative lysine methyltransferases (KMTs), in the C. parvum genome and classified them phylogenetically into distinct subfamilies (namely CpSET1, CpSET2, CpSET8, CpKMTox and CpAKMT). Our structural analysis further characterized CpSET1, CpSET2 and CpSET8 as histone lysine methyltransferases (HKMTs). The expression of the CpSET genes varies considerably during the parasite life cycle and specific methyl-lysine antibodies showed dynamic changes in parasite histone methylation during development (CpSET1:H3K4; CpSET2:H3K36; CpSET8:H4K20). We investigated the impact of C. parvum infection on the host histone lysine methylation. Remarkably, parasite infection led to a considerable decrease in host H3K36me3 and H3K27me3 levels, highlighting the potential of the parasite to exploit the host epigenetic regulation to its advantage. This is the first study to describe epigenetic mechanisms occurring throughout the parasite life cycle and during the host-parasite interaction. A better understanding of histone methylation in both parasite and host genomes may highlight novel infection control strategies.

Microarray analysis of circular RNAs in HCT-8 cells infected with Cryptosporidium parvum.

We read with great interest the article by Yin et al. (Parasit Vectors 14:238, 2021). The authors found that Cryptosporidium infection induced significantly aberrant expression of circular RNA profiles in HCT-8 cells, a finding which has far-reaching implications. However, due to the high number of false positives caused by multiple comparisons, statistical methods for microarray analysis should be carefully selected. Accurate analysis results will provide a convincing basis for subsequent experiments. In addition, we recommend several more appropriate methods in this article.

Serum IgG Responses to gp15 and gp40 Protein-Derived Synthetic Peptides From Cryptosporidium parvum.

Cryptosporidium spp. are responsible for moderate to severe diarrhea, mainly in children and immunocompromised patients. Using ELISA, the recognition of synthetic peptides generated from the sequences of the Cryptosporidium parvum gp40 and gp15 proteins by serum IgM and IgG antibodies from patients infected (cases) with Cryptosporidium hominis, C. parvum, and Cryptosporidium canis, and uninfected individuals (controls) was evaluated. A statistically significant difference (p = 0.0025) was found in terms of the recognition of peptides A133 and A32 between cases and controls. Additional studies are necessary to understand the potential of these peptides as vaccine candidates.

Reversal of Pathogen-Induced Barrier Defects in Intestinal Epithelial Cells by Contra-pathogenicity Agents.

BACKGROUND: Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa. AIMS: The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies. METHODS: Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response. RESULTS: All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-ß, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity. CONCLUSION: The enteropathogens impaired intestinal-epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity.

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

Cryptosporidium parvum upregulates miR-942-5p expression in HCT-8 cells via TLR2/TLR4-NF-κB signaling.

BACKGROUND: Micro (mi)RNAs are small noncoding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. This study investigated host miRNA activity in the innate immune response to Cryptosporidium parvum infection. METHODS: In vitro infection model adopts HCT-8 human ileocecal adenocarcinoma cells infected with C. parvum. The expression of miR-942-5p was estimated using quantitative real-time polymerase chain reaction (qPCR). The TLRs-NF-κB signaling was confirmed by qPCR, western blotting, TLR4- and TLR2-specific short-interfering (si)RNA, and NF-κB inhibition. RESULTS: HCT-8 cells express all known toll-like receptors (TLRs). Cryptosporidium parvum infection of cultured HCT-8 cells upregulated TLR2 and TLR4, and downstream TLR effectors, including NF-κB and suppressed IκBα (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha). The expression of miR-942-5p was significantly upregulated at 4, 8, 12 and 24 h post-infection, and especially at 8 hpi. The results of TLR4- and TLR2-specific siRNA and NF-κB inhibition showed that upregulation of miR-942-5p was promoted by p65 subunit-dependent TLR2/TLR4-NF-κB pathway signaling. CONCLUSIONS: miR-942-5p of HCT-8 cells was significantly upregulated after C. parvum infection, especially at 8 hpi, in response to a p65-dependent TLR2/TLR4-NF-κB signaling. TLR4 appeared to play a dominant role.

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites.

In humans, mRNA polyadenylation involves the participation of about 20 factors in four main complexes that recognize specific RNA sequences. Notably, CFIm25, CPSF73, and PAP have essential roles for poly(A) site selection, mRNA cleavage, and adenosine residues polymerization. Besides the relevance of polyadenylation for gene expression, information is scarce in intestinal protozoan parasites that threaten human health. To better understand polyadenylation in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum, which represent leading causes of diarrhea worldwide, genomes were screened for orthologs of human factors. Results showed that Entamoeba histolytica and C. parvum have 16 and 12 proteins out of the 19 human proteins used as queries, respectively, while G. lamblia seems to have the smallest polyadenylation machinery with only six factors. Remarkably, CPSF30, CPSF73, CstF77, PABP2, and PAP, which were found in all parasites, could represent the core polyadenylation machinery. Multiple genes were detected for several proteins in Entamoeba, while gene redundancy is lower in Giardia and Cryptosporidium. Congruently with their relevance in the polyadenylation process, CPSF73 and PAP are present in all parasites, and CFIm25 is only missing in Giardia. They conserve the functional domains and predicted folding of human proteins, suggesting they may have the same roles in polyadenylation.

In Vitro Culture of Cryptosporidium parvum Using Hollow Fiber Bioreactor: Applications for Simultaneous Pharmacokinetic and Pharmacodynamic Evaluation of Test Compounds.

Hollow fiber technology is a powerful tool for the culture of difficult-to-grow cells. Cryptosporidium parvum has a multistage sexual and asexual life cycle that has proved difficult to culture by conventional in vitro culture methods. Here, we describe a method utilizing a hollow fiber bioreactor for the continuous in vitro growth of C. parvum that produces sexual and asexual stages. The method enables the evaluation of potential therapeutic compounds under conditions that mirror the dynamic conditions found in the gut facilitating preliminary pharmacokinetic and pharmacodynamic data to be obtained.

Genetic ablation of purine salvage in Cryptosporidium parvum reveals nucleotide uptake from the host cell.

The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early-childhood mortality. Waterborne outbreaks occur frequently, even in countries with advanced water treatment capabilities, and there is currently no fully effective treatment. Nucleotide pathways are attractive targets for antimicrobial development, and several laboratories are designing inhibitors of these enzymes as potential treatment for Cryptosporidium infections. Here we take advantage of newly available molecular genetics for Cryptosporidium parvum to investigate nucleotide biosynthesis by directed gene ablation. Surprisingly, we found that the parasite tolerates the loss of classical targets including dihydrofolate reductase-thymidylate synthase (DHFR-TS) and inosine monophosphate dehydrogenase (IMPDH). We show that thymidine kinase provides a route to thymidine monophosphate in the absence of DHFR-TS. In contrast, only a single pathway has been identified for C. parvum purine nucleotide salvage. Nonetheless, multiple enzymes in the purine pathway, as well as the adenosine transporter, can be ablated. The resulting mutants are viable under normal conditions but are hypersensitive to inhibition of purine nucleotide synthesis in their host cell. Cryptosporidium might use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude that Cryptosporidium imports purine nucleotides from the host cell. The potential for ATP uptake from the host has significant impact on our understanding of parasite energy metabolism given that Cryptosporidium lacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle.

Attenuation of Intestinal Epithelial Cell Migration During Cryptosporidium parvum Infection Involves Parasite Cdg7_FLc_1030 RNA-Mediated Induction and Release of Dickkopf-1.

Intestinal infection by Cryptosporidium is known to cause epithelial cell migration disorder but the underlying mechanisms are unclear. Previous studies demonstrated that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using multiple models of intestinal cryptosporidiosis, we report here that C. parvum infection induces expression and release of the dickkopf protein 1 (Dkk1) from intestinal epithelial cells. Delivery of parasite Cdg7_FLc_1030 RNA to intestinal epithelial cells triggers transactivation of host Dkk1 gene during C. parvum infection. Release of Dkk1 is involved in C. parvum-induced inhibition of cell migration of epithelial cells, including noninfected bystander cells. Moreover, Dkk1-mediated suppression of host cell migration during C. parvum infection involves inhibition of Cdc42/Par6 signaling. Our data support the hypothesis that attenuation of intestinal epithelial cell migration during Cryptosporidium infection involves parasite Cdg7_FLc_1030 RNA-mediated induction and release of Dkk1 from infected cells.

Glycoproteins and Gal-GalNAc cause Cryptosporidium to switch from an invasive sporozoite to a replicative trophozoite.

The apicomplexan parasite Cryptosporidium causes cryptosporidiosis, a diarrheal disease that can become chronic and life threatening in immunocompromised and malnourished people. There is no effective drug treatment for those most at risk of severe cryptosporidiosis. The disease pathology is due to a repeated cycle of host cell invasion and parasite replication that amplifies parasite numbers and destroys the intestinal epithelium. This study aimed to better understand the Cryptosporidium replication cycle by identifying molecules that trigger the switch from invasive sporozoite to replicative trophozoite. Our approach was to treat sporozoites of Cryptosporidium parvum and Cryptosporidium hominis, the species causing most human cryptosporidiosis, with various media under axenic conditions and examine the parasites for rounding and nuclear division as markers of trophozoite development and replication, respectively. FBS had a concentration-dependent effect on trophozoite development in both species. Trophozoite development in C. parvum, but not C. hominis, was enhanced when RPMI supplemented with 10% FBS (RPMI-FBS) was conditioned by HCT-8 cells for 3h. The effect of non-conditioned and HCT-8 conditioned RPMI-FBS on trophozoite development was abrogated by proteinase K and sodium metaperiodate pretreatment, indicating a glycoprotein trigger. Cryptosporidium parvum and C. hominis trophozoite development also was triggered by Gal-GalNAc in a concentration-dependent manner. Cryptosporidium parvum replication was greatest following treatments with Gal-GalNAc, followed by conditioned RPMI-FBS and non-conditioned RPMI-FBS (P<0.05). Cryptosporidium hominis replication was significantly less than that in C. parvum for all treatments (P<0.05), and was greatest at the highest tested concentration of Gal-GalNAc (1mM).

Identification of invasion proteins of Cryptosporidium parvum.

Host cell interactions and invasion by Cryptosporidium is a complex process mediated by zoites ligand-host cell receptors. Knowledge of proteins involved in this process will enable entry level inhibitors to be tried as therapeutic agents. In the present study, invasion proteins of Cryptosporidium parvum were studied in vitro. Cryptosporidium sporozoites membrane proteins were isolated and Cy5 dye labelled. They were then allowed to interact with the intact host cells. The interacting proteins were identified using 2-dimensional gel electrophoresis followed by mass spectrometry analysis. Sixty-one proteins were identified including twenty-seven previously reported invasion proteins. The newly identified proteins such as serine/threonine protein kinase, PI4 kinase, Hsp105 and coiled coil may have their roles in the parasitic invasion process. Thus, a new approach was used in the study to identify the probable proteins involved in invasion and/or host-parasite interactions. The advantage of this method is that it takes only a months' time instead of decades to identify these proteins involved in invasion process.

Cryptosporidium parvum has an active hypusine biosynthesis pathway.

The protozoan parasite Cryptosporidium parvum causes severe enteric infection and diarrheal disease with substantial morbidity and mortality in untreated AIDS patients and children in developing or resource-limited countries. No fully effective treatment is available. Hypusination of eIF5A is an important post-translational modification essential for cell proliferation. This modification occurs in a two step process catalyzed by deoxyhypusine synthase (DHS) followed by deoxyhypusine hydroxylase. An ORF of 1086bp was identified in the C. parvum (Cp) genome which encodes for a putative polypeptide of 362 amino acids. The recombinant CpDHS protein was purified to homogeneity and used to probe the enzyme's mechanism, structure, and inhibition profile in a series of kinetic experiments. Sequence analysis and structural modeling of CpDHS were performed to probe differences with respect to the DHS of other species. Unlike Leishmania, Trypanosomes and Entamoeba, Cryptosporidium contains only a single gene for DHS. Phylogenetic analysis shows that CpDHS is more closely related to apicomplexan DHS than kinetoplastid DHS. Important residues that are essential for the functioning of the enzyme including NAD(+) binding residues, spermidine binding residues and the active site lysine are conserved between CpDHS and human DHS. N(1)-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor of DHS caused an effective inhibition of infection and growth of C. parvum in HCT-8 cells.

Characterization of a novel otubain-like cysteine protease of Cryptosporidium parvum.

Otubains are a recently discovered family of cysteine proteases that participate in the ubiquitin pathway. Here, we partially characterized the biochemical properties of a cysteine protease of Cryptosporidium parvum, which is closely related to otubains. The gene encoding otubain-like cysteine protease of C. parvum (CpOTU) contained the aspartate, cysteine and histidine residues that form the catalytic triad of otubains. The modified ubiquitin-associated domain and LxxL motif were identified in CpOTU. The recombinant CpOTU showed the isopeptidase activity at neutral pH values and its activity was effectively inhibited by ubiquitin aldehyde, N-ethylmaleimide and iodoacetic acid. Interestingly, CpOTU had an unusual C-terminal extension of 217 amino acids compared to mammalian otubains, and the C-terminal extension is essential for the activity of the enzyme. Expression of CpOTU peaked in the oocyst stage of the parasite, which suggested its potential physiological role for the oocyst stage.

Elongation factor-1α is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum.

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-ß- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

Cryptosporidium parvum induces an endoplasmic stress response in the intestinal adenocarcinoma HCT-8 cell line.

Invasion of human intestinal epithelial cells (HCT-8) by Cryptosporidium parvum resulted in a rapid induction of host cell spermidine/spermine N(1)-acetyltransferase 1 (hSSAT-1) mRNA, causing a 4-fold increase in SSAT-1 enzyme activity after 24 h of infection. In contrast, host cell SSAT-2, spermine oxidase, and acetylpolyamine oxidase (hAPAO) remained unchanged during this period. Intracellular polyamine levels of C. parvum-infected human epithelial cells were determined, and it was found that spermidine remained unchanged and putrescine increased by 2.5-fold after 15 h and then decreased after 24 h, whereas spermine decreased by 3.9-fold after 15 h. Concomitant with these changes, N(1)-acetylspermine and N(1)-acetylspermidine both increased by 115- and 24-fold, respectively. Increased SSAT-1 has previously been shown to be involved in the endoplasmic reticulum (ER) stress response leading to apoptosis. Several stress response proteins were increased in HCT-8 cells infected with C. parvum, including calreticulin, a major calcium-binding chaperone in the ER; GRP78/BiP, a prosurvival ER chaperone; and Nrf2, a transcription factor that binds to antioxidant response elements, thus activating them. However, poly(ADP-ribose) polymerase, a protein involved in DNA repair and programmed cell death, was decreased. Cumulatively, these results suggest that the invasion of HCT-8 cells by C. parvum results in an ER stress response by the host cell that culminates in overexpression of host cell SSAT-1 and elevated N(1)-acetylpolyamines, which can be used by a parasite that lacks ornithine decarboxylase.

Identification and characterization of Cryptosporidium parvum Clec, a novel C-type lectin domain-containing mucin-like glycoprotein.

Cryptosporidium species are waterborne apicomplexan parasites that cause diarrheal disease worldwide. Although the mechanisms underlying Cryptosporidium-host cell interactions are not well understood, mucin-like glycoproteins of the parasite are known to mediate attachment and invasion in vitro. We identified C. parvum Clec (CpClec), a novel mucin-like glycoprotein that contains a C-type lectin domain (CTLD) and has orthologs in C. hominis and C. muris. CTLD-containing proteins are ligand-binding proteins that function in adhesion and signaling and are present in a wide range of organisms, from humans to viruses. However, this is the first report of a CTLD-containing protein in protozoa and in Apicomplexa. CpClec is predicted to be a type 1 membrane protein, with a CTLD, an O-glycosylated mucin-like domain, a transmembrane domain, and a cytoplasmic tail containing a YXX sorting motif. The predicted structure of CpClec displays several characteristics of canonical CTLD-containing proteins, including a long loop region hydrophobic core associated with calcium-dependent glycan binding as well as predicted calcium- and glycan-binding sites. CpClec expression during C. parvum infection in vitro is maximal at 48 h postinfection, suggesting that it is developmentally regulated. The 120-kDa mass of native CpClec is greater than predicted, most likely due to O-glycosylation. CpClec is localized to the surface of the apical region and to dense granules of sporozoites and merozoites. Taken together, these findings, along with the known functions of C. parvum mucin-like glycoproteins and of CTLD-containing proteins, strongly implicate a significant role for CpClec in Cryptosporidium-host cell interactions.

Structure of Pseudomonas aeruginosa inosine 5'-monophosphate dehydrogenase.

Inosine 5'-monophosphate dehydrogenase (IMPDH) represents a potential antimicrobial drug target. The crystal structure of recombinant Pseudomonas aeruginosa IMPDH has been determined to a resolution of 2.25 Å. The structure is a homotetramer of subunits dominated by a (ß/α)8-barrel fold, consistent with other known structures of IMPDH. Also in common with previous work, the cystathionine ß-synthase domains, residues 92-204, are not present in the model owing to disorder. However, unlike the majority of available structures, clearly defined electron density exists for a loop that creates part of the active site. This loop, composed of residues 297-315, links α8 and ß9 and carries the catalytic Cys304. P. aeruginosa IMPDH shares a high level of sequence identity with bacterial and protozoan homologues, with residues involved in binding substrate and the NAD+ cofactor being conserved. Specific differences that have been proven to contribute to selectivity against the human enzyme in a study of Cryptosporidium parvum IMPDH are also conserved, highlighting the potential value of IMPDH as a drug target.