Voltage Gated Calcium Channels Negatively Regulate Protective Immunity to Mycobacterium tuberculosis

Mycobacterium tuberculosis modulates levels and activity of key intracellular second messengers to evade protective immune responses. Calcium release from voltage gated calcium channels (VGCC) regulates immune responses to pathogens. In this study, we investigated the roles of VGCC in regulating protective immunity to mycobacteria in vitro and in vivo. Inhibiting L-type or R-type VGCC in dendritic cells (DCs) either using antibodies or by siRNA increased calcium influx in an inositol 1,4,5-phosphate and calcium release calcium activated channel dependent mechanism that resulted in increased expression of genes favoring pro-inflammatory responses. Further, VGCC-blocked DCs activated T cells that in turn mediated killing of M. tuberculosis inside macrophages. Likewise, inhibiting VGCC in infected macrophages and PBMCs induced calcium influx, upregulated the expression of pro-inflammatory genes and resulted in enhanced killing of intracellular M. tuberculosis. Importantly, compared to healthy controls, PBMCs of tuberculosis patients expressed higher levels of both VGCC, which were significantly reduced following chemotherapy. Finally, blocking VGCC in vivo in M. tuberculosis infected mice using specific antibodies increased intracellular calcium and significantly reduced bacterial loads. These results indicate that L-type and R-type VGCC play a negative role in M. tuberculosis infection by regulating calcium mobilization in cells that determine protective immunity

Systematic deletion and site-directed mutagenesis of FHVB2 establish the role of C-terminal amino acid residues in RNAi suppression

Viruses and siRNA/miRNA machinery of the host cell interact in diverse ways with the virus encoded RNAi suppressor proteins. These interactions have implications on the replication and pathogenicity of the virus and also on the immune response of the host. Suppressor protein B2 of insect Flock House Virus (FHVB2), has been shown to mediate RNAi suppression via N-terminal region by directly binding to dsRNA. We have previously shown that FHVB2 protein also interacts with host Dicer protein via its PAZ domain. In the present study, we performed systematic mutagenesis studies to map the FHVB2 regions involved in mediating suppression of RNAi. Progressive deletion of 17 amino acids from N- and C-terminii of FHVB2 resulted in cumulative decrease in RNAi suppression activity of FHVB2. The deletion of 17 amino acids from the C-terminus resulted in more reduction in RNAi suppression in comparison to the N-terminal deletions. Subsequently, we generated 17 successive point mutants of FHVB2 C-terminus and evaluated the RNAi suppression activity for each of the point mutants. Each point mutation resulted in a significant reduction in RNAi suppression activity of FHVB2. These results provide evidence for the role of C-terminal of FHVB2 in RNAi suppression

Falcipain-1, a Plasmodium falciparum Cysteine Protease with Vaccine Potential

Cysteine proteases (falcipains) of Plasmodium falciparum are potential targets for antimalarial chemotherapy, since they have been shown to be involved in important cellular functions such as hemoglobin degradation and invasion/rupture of red blood cells during parasite life cycle. The role of falcipain-1 at the asexual blood stages of the parasite still remains uncertain. This is mainly due to a lack of methods to prepare this protein in an active form. In order to obtain biologically active falcipain-1, a number of falcipain-1 constructs were designed and a systematic assessment of the refolding conditions was done. We describe here the expression, purification, and characterization of a falcipain-1 construct encoding mature falcipain-1 and 35 amino acids from the C-terminal of the pro domain. Recombinant falcipain-1 was overexpressed in the form of inclusion bodies, solubilized, and purified by Ni(2+)-nitrilotriacetic acid affinity chromatography under denaturing conditions. A systemic approach was then followed to optimize refolding parameters. An optimum refolding condition was obtained, and the yield of the purified refolded falcipain-1 was ∼1 mg/liter. Activity of the protein was analyzed by fluorometric and gelatin degradation assays. Immunolocalization studies using anti-falcipain-1 sera revealed a distinct staining at the apical end of the P. falciparum merozoites. Previous studies using falcipain-1-specific inhibitors have suggested a role of falcipain-1 in merozoite invasion. Based on its localization and its role in invasion, we analyzed the immunogenicity of falcipain-1 in mice, followed by heterologous challenge with Plasmodium yoelii sporozoites. Our results suggest a possible role of falcipain-1 in merozoite invasion

Arrest of Nuclear Division in <em>Plasmodium</em> through Blockage of Erythrocyte Surface Exposed Ribosomal Protein P2

<div><p>Malaria parasites reside inside erythrocytes and the disease manifestations are linked to the growth inside infected erythrocytes (IE). The growth of the parasite is mostly confined to the trophozoite stage during which nuclear division occurs followed by the formation of cell bodies (schizogony). The mechanism and regulation of schizogony are poorly understood. Here we show a novel role for a <em>Plasmodium falciparum</em> 60S stalk ribosomal acidic protein P2 (PfP2) (PFC0400w), which gets exported to the IE surface for 6–8 hrs during early schizogony, starting around 26–28 hrs post-merozoite invasion. The surface exposure is demonstrated using multiple PfP2-specific monoclonal antibodies, and is confirmed through transfection using PfP2-GFP. The IE surface-exposed PfP2-protein occurs mainly as SDS-resistant P2-homo-tetramers. Treatment with anti-PfP2 monoclonals causes arrest of IEs at the first nuclear division. Upon removal of the antibodies, about 80–85% of synchronized parasites can be released even after 24 hrs of antibody treatment. It has been reported that a tubovesicular network (TVN) is set up in early trophozoites which is used for nutrient import. Anti-P2 monoclonal antibodies cause a complete fragmentation of TVN by 36 hrs, and impairs lipid import in IEs. These may be downstream causes for the cell-cycle arrest. Upon antibody removal, the TVN is reconstituted, and the cell division progresses. Each of the above properties is observed in the rodent malaria parasite species <em>P. yoelii</em> and <em>P. berghei</em>. The translocation of the P2 protein to the IE surface is therefore likely to be of fundamental importance in <em>Plasmodium</em> cell division.</p> </div

Immunoblot, mass spectrometry, flow cytometry and IFA of parasite and RBC localized PfP2 protein.

<p>Infected erythrocytes were lysed gently with saponin to separate parasite and the IE components. <b>A:</b> Synchronized <i>P. falciparum</i> parasites were harvested at various time points post-merozoite invasion (PMI) during the development of parasite in erythrocytes. Immunoblots with 40 µg of total parasite protein preparation (I and II) and 80 µg of IE ghost and IE cytosol (IV) were probed with various antibodies. IE ghost and cytosol at various time points are depicted as G and C lanes, respectively. Panel III shows a histogram of fluctuation of parasite P2 protein intensity averaged over three independent experiments, normalized to β-actin levels. Panel V shows the total parasite protein concentration at each time point PMI. <b>B:</b> Coomassie stain and immunoblot of infected erythrocyte (IE) ghost (40 µg) and uninfected erythrocyte (UIE) ghosts (120 µg) resolved on SDS-PAGE, probed with E2G12. The arrow in the Coomassie stained gel (65 kDa region) was cut out and used for Mass spectrometric analysis using Electronspray Ionization Mass spectrometry/Mass spectrometry (ESI/MS/MS) which yielded the peptide MAMK of P2 protein (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002858#ppat.1002858.s012" target="_blank">Table S1</a>). <b>C:</b> Flow cytometric data on population of cells positive for IE surface exposed P2 using E2G12 across various stages of <i>P. falciparum</i>. <b>D:</b> Flow cytometric analysis of IEs using DAPI across the 48 hrs life cycle of <i>P. falciparum</i>. For analysis, total 6 million cells were counted. The statistical comparison for each data point is made with the corresponding control (no antibody) value. * <i>P</i><0.05, ** <i>P</i><0.01. <b>E</b>: IFA of synchronized <i>P. falciparum</i> cells using DAPI (blue), P2 (green), and bright field of IE at various time points PMI in parasite development. Scale bar indicates 2 µm.</p

Immunofluorescence assay of <i>P. falciparum</i> infected erythrocytes (IE) using various antibodies.

<p>(<b>A</b>) & (<b>B</b>) show confocal images of permiabilized (IFA) and unpermeabilized in solution (SIFA) assays respectively, of different IE stages (I, II: Ring; III, IV, V, VII, VIII, IX: trophozoite; VI: schizont) probed with I-VI: anti-PfP2 mAbs. In all cases mAbs E2G12 was used except panels A V and B IV, V where A12D9 was used. VII: antiPfP1 polyclonal; VIII: anti-PfP0 mAbs E5F4; IX: Phalloidin (for staining β-actin). In each panel the IE nucleus/nuclei were stained using DAPI (first column), antibody staining (second column), and a merge of antibody with bright field (third column). (<b>C</b>) Localization of DAPI (blue), P2 (green); various antibodies (anti-PfP1, anti β-tubulin, anti-PfP0, and anti-MSP1) in red at the di-nuclear (DN) stage. R: Ring; T: Trophozoite; S: Schizont; SN: single nucleus; DN: Di-nuclear; MN: Multi-nuclear stages of <i>Plasmodium falciparum</i> infected erythrocytes. Scale bar indicates 2 µm.</p