Identification and Characterization of a Novel Calcium-Activated Apyrase from Cryptosporidium Parasites and Its Potential Role in Pathogenesis

Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, United States of America.
PLoS ONE (Impact Factor: 3.23). 02/2012; 7(2):e31030. DOI: 10.1371/journal.pone.0031030
Source: PubMed


Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.

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Available from: Patricio Manque
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    • "An ability to cleave ATP and/or ADP in the extracellular environment has been described in several pathogens. For example ecto-ATPase activity has been described in the protozoan parasites Toxoplasma gondii (Bermudes et al., 1994), Leishmania amazonensis (Berredo-Pinho et al., 2001), Trichomonas vaginalis (de Jesus et al., 2002) and Cryptosporidum parvum (Manque et al., 2012) and in several bacterial pathogens including Mycobacterium bovis (Zaborina et al., 1999), Vibrio cholera (Punj et al., 2000), Staphylococcus aureus (Thammavongsa et al., 2009), and Legionella pneumophilia (Vivian et al., 2010). Similarly, blood-feeding ectoparasites are known to release a repertoire of nucleotide-metabolizing enzymes in their saliva (Andersen et al., 2007; de Araujo et al., 2012). "
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    ABSTRACT: Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs) and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP). Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5) and ATP diphosphohydrolase (SmATPDase1). In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms' ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ± 0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals like ATP, and pro-thrombotic signals like ADP, these parasite enzymes may minimize host immune responses, inhibit blood coagulation and promote schistosome survival.
    Full-text · Article · Mar 2014 · PeerJ
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    • "Thus, an intra-nasal prime-boost vaccine regimen is now available for eliciting a sustained systemic as well as a local response to specific Cryptosporidial antigens, irrespective of the nutritional status of the host. Application of this methodology to putative disease-protective antigens such as a calcium-activated apyrase for cryptosporidiosis [35], may be indicated. "
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    ABSTRACT: Cryptosporidium is a protozoan parasite associated with acute and persistent diarrhea that, even in asymptomatic persons, can impair normal growth and potentially cognitive and physical development in young children. The recent availability of the complete gene sequence for Cryptosporidium hominis antigen Cp15 allows examination of innovative vaccine regimens involving intra-nasal antigen priming with live bacterial vectors applicable to human populations. We used a recently described weaned mouse model of cryptosporidiosis, where nourished and malnourished vaccinated mice receive the Cp15 antigen recombinant with cytolysinA on a Salmonella serovar Typhi CVD 908-htr A vector, followed by parenteral exposure to antigen with adjuvant. After challenge with Cryptosporidium oocysts via gavage, parameters of infection and disease (stool shedding of parasites, growth rates) were quantified, and serum/lymphoid tissue harvested to elucidate the Cp15-specific adaptive immune response. In vaccinated nourished mice, the regimen was highly immunogenic, with strong antigen-specific IL-6 and IFN-γ secretion and robust Cp15-specific immunoglobulin titers. In vaccinated malnourished mice, secretion of cytokines, particularly IFN-γ, and antigen-specific humoral immunity were generally undiminished despite protein deprivation and stunted growth. In contrast, after natural (oral) challenge with an identical inoculum of Cryptosporidium oocysts, cytokine and humoral responses to Cp15 were less than one-fourth those in vaccinated mice. Nevertheless, vaccination resulted in only transient reduction in stool shedding of parasites and was not otherwise protective against disease. Overall, immunogenicity for a C. hominis antigen was documented in mice, even in the setting of prolonged malnutrition, using an innovative vaccine regimen involving intra-nasal antigen priming with a live enteric bacterial vector, that has potential applicability to vulnerable human populations irrespective of nutritional status.
    Full-text · Article · Dec 2012 · Vaccine
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    No preview · Article · Aug 2013 · Zoonoses and Public Health
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