Leishmania infantum nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) B-domain: Antibody antiproliferative effect on the promastigotes and IgG subclass responses in canine visceral leishmaniasis.

A nucleoside triphosphate diphosphohydrolase-1 (NTPDase 1) was identified on the surface, flagellum and kinetoplast from L. infantum promastigotes by immunocytochemistry and confocal laser scanning microscopy, using immune sera that recognized specifically the B domain of NTPDase 1 and produced against synthetic peptides (LbB1LJ and LbB2LJ) derived from this domain. The polyclonal antibodies had effective antileishmanial effect, reducing significantly in vitro promastigotes growth (21-25%), an antiproliferative effect also demonstrated by immune sera produced against recombinant r-pot B domain, and two other synthetic peptides (potB1LJ and potB2LJ). In addition, using these biomolecules in ELISA technique, IgG1 and IgG2 subclasses reactivities of either healthy dogs or infected by L. infantum and classified clinically as asymptomatic, oligosymptomatic and symptomatic were tested. Analysis of distinct IgG1 and IgG2 seropositivities patterns suggested antibody subclasses binding epitopes along B domain for protection against infection, indicating this domain as a new tool for prophylactic and immunotherapeutic investigations.

Subcellular Localization and Functional Characterization of GII.4 Norovirus-Encoded NTPase.

The genotype II.4 (GII.4) variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded nonstructural proteins (including Nterm, NTPase, P22, VPg, Pro, and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (designated GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or nonvesicular textures in the cell cytoplasm, and the nonvesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. In particular, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the GI HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as with Nterm and P22, but not VPg, Pro, and RdRp, in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail, or head-to-tail configuration. More importantly, we demonstrate that GII-NTPase possesses a proapoptotic activity, which can be further enhanced by coexpression with Nterm or P22.IMPORTANCE Despite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral nonstructural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a GI strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate (i) vesicle formation, (ii) ER colocalization, (iii) the interaction with two other nonstructural proteins, including Nterm and P22, (iv) the formation of homodimers or homo-oligomers, and (v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.

Germ-Cell-Specific Inflammasome Component NLRP14 Negatively Regulates Cytosolic Nucleic Acid Sensing to Promote Fertilization.

Cytosolic sensing of nucleic acids initiates tightly regulated programs to limit infection. Oocyte fertilization represents a scenario wherein inappropriate responses to exogenous yet non-pathogen-derived nucleic acids would have negative consequences. We hypothesized that germ cells express negative regulators of nucleic acid sensing (NAS) in steady state and applied an integrated data-mining and functional genomics approach to identify a rheostat of DNA and RNA sensing-the inflammasome component NLRP14. We demonstrated that NLRP14 interacted physically with the nucleic acid sensing pathway and targeted TBK1 (TANK binding kinase 1) for ubiquitination and degradation. We further mapped domains in NLRP14 and TBK1 that mediated the inhibitory function. Finally, we identified a human nonsense germline variant associated with male sterility that results in loss of NLRP14 function and hyper-responsiveness to nucleic acids. The discovery points to a mechanism of nucleic acid sensing regulation that may be of particular importance in fertilization.

Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1.

SAMHD1 is a host protein responsible, at least in part, for the inefficient infection of dendritic, myeloid, and resting T cells by HIV-1. Interestingly, HIV-2 and SIVsm viruses are able to counteract SAMHD1 by targeting it for proteasomal degradation using their Vpx proteins. It has been proposed that SAMHD1 is a dGTP-dependent deoxynucleoside triphosphohydrolase (dNTPase) that restricts HIV-1 by reducing cellular dNTP levels to below that required for reverse transcription. However, nothing is known about SAMHD1 posttranslational modifications and their potential role in regulating SAMHD1 function. We used (32)P labeling and immunoblotting with phospho-specific antibodies to identify SAMHD1 as a phosphoprotein. Several amino acids in SAMHD1 were identified to be sites of phosphorylation using direct mass spectrometry. Mutation of these residues to alanine to prevent phosphorylation or to glutamic acid to mimic phosphorylation had no effect on the nuclear localization of SAMHD1 or its sensitivity to Vpx-mediated degradation. Furthermore, neither alanine nor glutamic acid substitutions had a significant effect on SAMHD1 dNTPase activity in an in vitro assay. Interestingly, however, we found that a T592E mutation, mimicking constitutive phosphorylation at a main phosphorylation site, severely affected the ability of SAMHD1 to restrict HIV-1 in a U937 cell-based restriction assay. In contrast, a T592A mutant was still capable of restricting HIV-1. These results indicate that SAMHD1 phosphorylation may be a negative regulator of SAMHD1 restriction activity. This conclusion is supported by our finding that SAMHD1 is hyperphosphorylated in monocytoid THP-1 cells under nonrestrictive conditions.

Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II.

The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain (P<0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II+alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii.

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.

[Application of double antibody sandwich ELISA for detection of nucleoside triphosphate hydrolase-II protein of Toxoplasma gondii].

OBJECTIVE: To establish a double antibody sandwich ELISA method for detection of nucleoside triphosphate hydrolase-II (NTPase-II) protein of Toxoplasma gondii. METHODS: BALB/c mice were immunized with recombinant NTPase-II (rTgNTPase-II) protein of T. gondii. The hybridomas that secreted high titer of monoclonal antibodies (mAbs) with high specificity were screened and used to establish the double antibody sandwich ELISA for the detection of rTgNTPase-II. In order to evaluate the sensitivity of the method, the concentration of whole-tachyzoite lysate and rTgNTPase-II was detected, respectively. Serum samples from patients with malaria (7 cases), schistosomiasis (12 cases), paragonimiasis (14 cases) and cysticercosis (10 cases) were examined by the same method. RESULTS: Two hybridoma cell lines, MNTI and MNT2, were developed for secreting mAbs against rTgNTPase-II. Western blotting analysis showed that the two mAbs specifically recognized rTgNTPase-II and whole-tachyzoite lysate. The MNT1 was used as coating antibody, and HRP-labeled MNT2 as secondary antibody. The double antibody sandwich ELISA detecting rTgNTPase-II was developed with a minimum concentration of 6 microg/ml for whole-tachyzoite lysate and 1.5 microg/ml for rTgNTPase-II. An overall specificity of 100% was determined. CONCLUSION: The double antibody sandwich ELISA based on MNT1 as coating antibody and MNT2 as secondary antibody has a high specificity.

[Expression and antigenicity analysis of NTPase gene of Toxoplasma gondii].

OBJECTIVE: To clone and express prokaryotic recombinant plasmid of nucleoside triphosphate hydrolase (NTPase) gene of Toxoplasma gondii, and analyze its antigenicity. METHOD: NTPase gene was amplified by PCR from RH strain of T. gondii and cloned into pGEM-T Easy vector. Positive clones were screened and identified by BglII, HindIII digestion and sequenced. The target gene was then subcloned into prokaryotic expression vector pBAD-HisB and transformed into E. coli BL21 (DE3). The expressed recombinant protein was purified with Ni-NTA agarose and further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: NTPase-II gene was specifically amplified, and the homology of DNA sequence was 100% to that in the GenBank. SDS-PAGE showed that the recombinant NTPase protein with correct molecular weight was expressed highly in E.coli BL21 (DE3). Western blotting testified that the purified recombinant protein could be specifically recognized by mouse serum immunized with T. gondii and mouse anti-recombinant protein serum. CONCLUSION: The NTPase-II gene has been cloned and expressed in E.coli BL21 (DE3), and the purified protein of NTPase-II gene displays a specific antigenicity.