Interplay between transcriptional regulators and the SAGA chromatin modifying complex fine-tune iron homeostasis.

The human fungal pathogen Candida albicans responds to iron deprivation by a global transcriptome reconfiguration known to be controlled by the transcriptional regulators Hap43 (also known as Cap2), Sef1, and the trimeric Hap2-Hap3-Hap5 complex. However, the relative roles of these regulators are not known. To dissect this system, we focused on the FRP1 and ACO1 genes, which are induced and repressed, respectively, under iron deprivation conditions. Chromatin immunoprecipitation assays showed that the trimeric HAP complex and Sef1 are recruited to both FRP1 and ACO1 promoters. While the HAP complex occupancy at the FRP1 promoter was Sef1-dependent, occupancy of Sef1 was not dependent on the HAP complex. Furthermore, iron deprivation elicited histone H3-Lys9 hyperacetylation and Pol II recruitment mediated by the trimeric HAP complex and Sef1 at the FRP1 promoter. In contrast, at the ACO1 promoter, the HAP trimeric complex and Hap43 promoted histone deacetylation and also limited Pol II recruitment under iron deprivation conditions. Mutational analysis showed that the SAGA subunits Gcn5, Spt7, and Spt20 are required for C. albicans growth in iron-deficient medium and for H3-K9 acetylation and transcription from the FRP1 promoter. Thus, the trimeric HAP complex promotes FRP1 transcription by stimulating H3K9Ac and Pol II recruitment and, along with Hap43, functions as a repressor of ACO1 by maintaining a deacetylated promoter under iron-deficient conditions. Thus, a regulatory network involving iron-responsive transcriptional regulators and the SAGA histone modifying complex functions as a molecular switch to fine-tune tight control of iron homeostasis gene expression in C. albicans.

Aedes aegypti lachesin protein binds to the domain III of envelop protein of Dengue virus-2 and inhibits viral replication.

Dengue virus (DENV) comprises of four serotypes (DENV-1 to -4) and is medically one of the most important arboviruses (arthropod-borne virus). DENV infection is a major human health burden and is transmitted between humans by the insect vector, Aedes aegypti. Ae. aegypti ingests DENV while feeding on infected humans, which traverses through its gut, haemolymph and salivary glands of the mosquito before being injected into a healthy human. During this process of transmission, DENV must interact with many proteins of the insect vector, which are important for its successful transmission. Our study focused on the identification and characterisation of interacting protein partners in Ae. aegypti to DENV. Since domain III (DIII) of envelope protein (E) is exposed on the virion surface and is involved in virus entry into various cells, we performed phage display library screening against domain III of the envelope protein (EDIII) of DENV-2. A peptide sequence showing similarity to lachesin protein was found interacting with EDIII. The lachesin protein was cloned, heterologously expressed, purified and used for in vitro interaction studies. Lachesin protein interacted with EDIII and also with DENV. Further, lachesin protein was localised in neuronal cells of different organs of Ae. aegypti by confocal microscopy. Blocking of lachesin protein in Ae. aegypti with anti-lachesin antibody resulted in a significant reduction in DENV replication.

Myg1 exonuclease couples the nuclear and mitochondrial translational programs through RNA processing.

Semi-autonomous functioning of mitochondria in eukaryotic cell necessitates coordination with nucleus. Several RNA species fine-tune mitochondrial processes by synchronizing with the nuclear program, however the involved components remain enigmatic. In this study, we identify a widely conserved dually localized protein Myg1, and establish its role as a 3'-5' RNA exonuclease. We employ mouse melanoma cells, and knockout of the Myg1 ortholog in Saccharomyces cerevisiae with complementation using human Myg1 to decipher the conserved role of Myg1 in selective RNA processing. Localization of Myg1 to nucleolus and mitochondrial matrix was studied through imaging and confirmed by sub-cellular fractionation studies. We developed Silexoseqencing, a methodology to map the RNAse trail at single-nucleotide resolution, and identified in situ cleavage by Myg1 on specific transcripts in the two organelles. In nucleolus, Myg1 processes pre-ribosomal RNA involved in ribosome assembly and alters cytoplasmic translation. In mitochondrial matrix, Myg1 processes 3'-termini of the mito-ribosomal and messenger RNAs and controls translation of mitochondrial proteins. We provide a molecular link to the possible involvement of Myg1 in chronic depigmenting disorder vitiligo. Our study identifies a key component involved in regulating spatially segregated organellar RNA processing and establishes the evolutionarily conserved ribonuclease as a coordinator of nucleo-mitochondrial crosstalk.

Deciphering the role of calcium homeostasis in T cells functions during mycobacterial infection.

Calcium plays an important role in regulating cell physiology and immune responses to various pathogens. Our recent work has highlighted the crucial role for calcium homeostasis in dendritic cells and macrophages during various infections. Here we investigated the effect of calcium homeostasis in regulating T cell activation and function during mycobacterial infection. Results show that calcium homeostasis had varied effects in regulating T cell activation and function during mycobacterial infection. This included regulation of the expression of co-stimulatory molecules, cytokine profiles and effector function. A net negative role for Voltage Gated Calcium Channel (VGCC) was observed. Inhibiting VGCC in mycobacteria primed T cells induced increased production of pro-inflammatory cytokines and an increased effector phenotype. Infected macrophages when incubated with VGCC inhibited T cells, induced increased expression of co-stimulatory molecule expression on macrophages, increased the production of pro-inflammatory cytokines and increased autophagy and apoptosis. This collectively led to reduced survival of mycobacteria inside macrophages. The data point towards a fine regulation of protective responses by routes of calcium influx and release that mediate pathogen survival or clearance.

Biochemical characterization of Plasmodium complement factors binding protein for its role in immune modulation.

Complement system is the first line of human defence against intruding pathogens and is recognized as a potentially useful therapeutic target. Human malaria parasite Plasmodium employs a series of intricate mechanisms that enables it to evade different arms of immune system, including the complement system. Here, we show the expression of a multi-domain Plasmodium Complement Control Protein 1, PfCCp1 at asexual blood stages and its binding affinity with C3b as well as C4b proteins of human complement cascade. Using a biochemical assay, we demonstrate that PfCCp1 binds with complement factors and inhibits complement activation. Active immunization of mice with PfCCp1 followed by challenge with Plasmodium berghei resulted in the loss of biphasic growth of parasites and early death in comparison to the control group. The study also showed a role of PfCCp1 in modulating Toll-like receptor (TLR)-mediated signalling and effector responses on antigen-presenting cells. PfCCp1 binds with dendritic cells that down-regulates the expression of signalling molecules and pro-inflammatory cytokines, thereby dampening the TLR2-mediated signalling; hence acting as a potent immuno-modulator. In summary, PfCCp1 appears to be an important component of malaria parasite directed immuno-modulating strategies that promote the adaptive fitness of pathogens in the host.

Functional specialization of two paralogous TAF12 variants by their selective association with SAGA and TFIID transcriptional regulatory complexes.

TATA box-binding protein (TBP)-associated factors (TAFs), evolutionarily conserved from yeast to humans, play a central role during transcription initiation. A subset of TAF proteins is shared in transcription factor II D (TFIID) and SAGA transcription regulatory complexes. Although higher eukaryotes contain multiple TAF variants that specify tissue- and developmental stage-specific organization of TFIID or SAGA complexes, in unicellular genomes, however, each TAF is encoded by a single gene. Surprisingly, we found that the genome of Candida albicans, the predominant human fungal pathogen, contains two paralogous TAF12 genes, CaTAF12L and CaTAF12, encoding H2B-like histone-fold domain-containing variants. Of the available fungal genome sequences, only seven other closely related diploid pathogenic Candida genomes encode the two TAF12 paralogs. Using affinity purifications from C. albicans cell extracts, we demonstrate that CaTAF12L uniquely associates with the SAGA complex and CaTAF12 associates with the TFIID complex. We further show that CaTAF12, but not CaTAF12L, is essential for C. albicans growth. Conditional depletion of the two TAF12 variant proteins caused distinct cellular and colony phenotypes. Together our results define a specialized organization of the TAF12 variants and non-redundant roles for the two TAF12 variants in the unicellular C. albicans genome.

Set2 methylation of histone H3 lysine 36 suppresses histone exchange on transcribed genes.

Set2-mediated methylation of histone H3 at Lys 36 (H3K36me) is a co-transcriptional event that is necessary for the activation of the Rpd3S histone deacetylase complex, thereby maintaining the coding region of genes in a hypoacetylated state. In the absence of Set2, H3K36 or Rpd3S acetylated histones accumulate on open reading frames (ORFs), leading to transcription initiation from cryptic promoters within ORFs. Although the co-transcriptional deacetylation pathway is well characterized, the factors responsible for acetylation are as yet unknown. Here we show that, in yeast, co-transcriptional acetylation is achieved in part by histone exchange over ORFs. In addition to its function of targeting and activating the Rpd3S complex, H3K36 methylation suppresses the interaction of H3 with histone chaperones, histone exchange over coding regions and the incorporation of new acetylated histones. Thus, Set2 functions both to suppress the incorporation of acetylated histones and to signal for the deacetylation of these histones in transcribed genes. By suppressing spurious cryptic transcripts from initiating within ORFs, this pathway is essential to maintain the accuracy of transcription by RNA polymerase II.

IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation.

Cellular homeostasis is an outcome of complex interacting processes with nonlinear feedbacks that can span distinct spatial and temporal dimensions. Skin tanning is one such dynamic response that maintains genome integrity of epidermal cells. Although pathways underlying hyperpigmentation cascade are recognized, negative feedback regulatory loops that can dampen the activated melanogenesis process are not completely understood. In this study, we delineate a regulatory role of IFN-γ in skin pigmentation biology. We show that IFN-γ signaling impedes maturation of the key organelle melanosome by concerted regulation of several pigmentation genes. Withdrawal of IFN-γ signal spontaneously restores normal cellular programming. This effect in melanocytes is mediated by IFN regulatory factor-1 and is not dependent on the central regulator microphthalmia-associated transcription factor. Chronic IFN-γ signaling shows a clear hypopigmentation phenotype in both mouse and human skin. Interestingly, IFN-γ KO mice display a delayed recovery response to restore basal state of epidermal pigmentation after UV-induced tanning. Together, our studies delineate a new spatiotemporal role of the IFN-γ signaling network in skin pigmentation homeostasis, which could have implications in various cutaneous depigmentary and malignant disorders.

A CUG codon-adapted anchor-away toolkit for functional analysis of genes in Candida albicans.

Promoter shutoff of essential genes in the diploid Candida albicans has often been insufficient to create tight, conditional null alleles due to leaky expression and has been a stumbling block in pathogenesis research. Moreover, homozygous deletion of non-essential genes has often been problematic due to the frequent aneuploidy in the mutant strains. Rapid, conditional depletion of essential genes by the anchor-away strategy has been successfully employed in Saccharomyces cerevisiae and other model organisms. Here, rapamycin mediates the dimerization of human FK506-binding protein (FKBP12) and FKBP12-rapamycin-binding (FRB) domain-containing target protein, resulting in relocalization to altered sub-cellular locations. In this work, we used the ribosomal protein Rpl13 as the anchor and took two nuclear proteins as targets to construct a set of mutants in a proof-of-principle approach. We first constructed a rapamycin-resistant C. albicans strain by introducing a dominant mutation in the CaTOR1 gene and a homozygous deletion of RBP1, the ortholog of FKBP12, a primary target of rapamycin. The FKBP12 and the FRB coding sequences were then CUG codon-adapted for C. albicans by site-directed mutagenesis. Anchor-away strains expressing the essential TBP1 gene or the non-essential SPT8 gene as FRB fusions were constructed. We found that rapamycin caused rapid cessation of growth of the TBP-AA strain within 15 minutes and the SPT8-AA strain phenocopied the constitutive filamentous phenotype of the spt8Δ/spt8Δ mutant. Thus, the anchor-away toolbox for C. albicans developed here can be employed for genome-wide analysis to identify gene function in a rapid and reliable manner, further accelerating anti-fungal drug development in C. albicans. IMPORTANCE: Molecular genetic studies thus far have identified ~27% open-reading frames as being essential for the vegetative growth of Candida albicans in rich medium out of a total 6,198 haploid set of open reading frames. However, a major limitation has been to construct rapid conditional alleles of essential C. albicans genes with near quantitative depletion of encoded proteins. Here, we have developed a toolbox for rapid and conditional depletion of genes that would aid studies of gene function of both essential and non-essential genes.

Butyrate induces oxidative burst mediated apoptosis via Glucose-6-Phosphate Dehydrogenase (G6PDH) in macrophages during mycobacterial infection.

Microorganisms present in the gut modulate host defence responses against infections in order to maintain immune homeostasis. This host-microbe crosstalk is regulated by gut metabolites. Butyrate is one such small chain fatty acid produced by gut microbes upon fermentation that has the potential to influence immune responses. Here we investigated the role of butyrate in macrophages during mycobacterial infection. Results demonstrate that butyrate significantly suppresses the growth kinetics of mycobacteria in culture medium as well as inhibits mycobacterial survival inside macrophages. Interestingly, butyrate alters the pentose phosphate pathway by inducing higher expression of Glucose-6-Phosphate Dehydrogenase (G6PDH) resulting in a higher oxidative burst via decreased Sod-2 and increased Nox-2 (NADPH oxidase-2) expression. Butyrate-induced G6PDH also mediated a decrease in mitochondrial membrane potential. This in turn lead to an induction of apoptosis as measured by lower expression of the anti-apoptotic protein Bcl-2 and a higher release of Cytochrome C as a result of induction of apoptosis. These results indicate that butyrate alters the metabolic status of macrophages and induces protective immune responses against mycobacterial infection.

Potassium ion channel Kir2.1 negatively regulates protective responses to Mycobacterium bovis BCG.

Tuberculosis caused by the pathogen Mycobacterium tuberculosis leads to increased mortality and morbidity worldwide. The prevalence of highly drug resistant strains has reinforced the need for greater understanding of host-pathogen interactions at the cellular and molecular levels. Our previous work demonstrated critical roles of calcium ion channels in regulating protective responses to mycobacteria. In this report we deciphered the roles of inwardly rectifying K+ ion channel Kir2.1 in epithelial cells. Data showed that infection of epithelial cells (and macrophages) increases the surface expression of Kir2.1. This increased expression of Kir2.1 results in higher intracellular mycobacterial survival, since either inhibiting or knocking down Kir2.1 results in mounting of a higher oxidative burst leading to a significant attenuation of mycobacterial survival. Further, inhibiting Kir2.1 also led to increased expression of T cell costimulatory molecules accompanied with increased activation of MAP Kinases and transcription factors NF-κB and pCREB. Furthermore, inhibiting Kir2.1 induced increased autophagy and apoptosis that could also contribute to decreased bacterial survival. Interestingly, an increased association of heat shock protein-70 with Kir2.1 was observed. The above results showed that mycobacteria modulate the expression and function of Kir2.1 in epithelial cells to its advantage.

Suppression of dendritic cell-mediated responses by genes in calcium and cysteine protease pathways during Mycobacterium tuberculosis infection.

With rising incidence of acquired drug resistance among life-threatening pathogens, alternative approaches to improve therapy and vaccination have taken center stage. To this end, genome-wide and pathway-specific siRNA libraries are being employed increasingly to identify genes that regulate immune responses against a number of pathogens. In this study using calcium and cysteine protease pathway-specific siRNA libraries, we identified genes that play critical roles in modulating diverse functions of dendritic cells (DCs) during Mycobacterium tuberculosis infection. Knockdown of many of these genes in the two pathways resulted in reduced bacterial burden within DCs. These included genes that regulated activation of transcription factors, ubiquitin-specific peptidases, and genes that are involved in autophagy and neddylation. Knockdown of certain genes increased the expression of IL-12p40 and surface densities of costimulatory molecules in an antigen- and receptor-specific manner. Increased IL-12p40 and costimulatory molecules on DCs also promoted the development of Th1 responses from a Th2 inducing antigen. Furthermore, modulation of autophagy and oxidative burst appeared to be one of the mechanisms by which these genes regulated survival of M. tuberculosis within DCs. Although some genes regulated specific responses, others regulated multiple responses that included IL-12 production, T cell priming, as well as intracellular survival of M. tuberculosis. Further dissection of the mechanisms such as neddylation, by which these genes regulate immune responses, would improve our understanding of host parameters that are modulated during M. tuberculosis infection.

HSP-27 and HSP-70 negatively regulate protective defence responses from macrophages during mycobacterial infection.

Mycobacterium tuberculosis attenuates many defence responses from alveolar macrophages to create a niche at sites of infection in the human lung. Levels of Heat Shock Proteins have been reported to increase many folds in the serum of active TB patients than in latently infected individuals. Here we investigated the regulation of key defence responses by HSPs during mycobacterial infection. We show that infection of macrophages with M. bovis BCG induces higher expression of HSP-27 and HSP-70. Inhibiting HSP-27 and HSP-70 prior to mycobacterial infection leads to a significant decrease in mycobacterial growth inside macrophages. Further, inhibiting HSPs resulted in a significant increase in intracellular oxidative burst levels. This was accompanied by an increase in the levels of T cell activation molecules CD40 and IL-12 receptor and a concomitant decrease in the levels of T cell inhibitory molecules PD-L1 and IL-10 receptor. Furthermore, inhibiting HSPs significantly increased the expression of key proteins in the autophagy pathway along with increased activation of pro-inflammatory promoting transcription factors NF-κB and p-CREB. Interestingly, we also show that both HSP-27 and HSP-70 are associated with anti-apoptotic proteins Bcl-2 and Beclin-1. These results point towards a suppressive role for host HSP-27 and HSP-70 during mycobacterial infection.

Mycobacteria modulate SUMOylation to suppresses protective responses in dendritic cells.

Post translational modifications (PTMs) are exploited by various pathogens in order to escape host immune responses. SUMOylation is one of the PTMs which is involved in regulation of a variety of cellular responses. However, the effects of host SUMOylation on pathogenic bacteria largely remain elusive. We, therefore, investigated the role of SUMOylation in regulating defense responses in dendritic cells (DCs) during mycobacterial infection. Dendritic Cells of female BALB/c mice and THP-1 macrophages were used. Western blotting was performed to measure the expression of level of SUMO1, pSTAT1, pp38, pERK, Beclin-1, LC3, Bax and Cytochrome C. For bacterial burden confocal microscopy and CFU (Colony Forming Unit) were used. Flow cytometry was used for ROS and co-stimulatory molecules measurement. Cytokine level were measured using ELISA. We show that stimulation of Bone Marrow Derived Dendritic Cells (BMDCs) with mycobacterial antigen Rv3416 or live infection with Mycobacterium bovis BCG increases the SUMOylation of host proteins. Inhibition of SUMOylation significantly decreased intracellular bacterial loads in DCs. Additionally, inhibiting SUMOylation, induces protective immune responses by increasing oxidative burst, pro-inflammatory cytokine expression and surface expression of T cell co-stimulatory molecules, and activation of pSTAT1 and Mitogen Activated Protein Kinases (MAPK) proteins- pp38 and pERK. SUMOylation inhibition also increased apoptosis and autophagy in BMDCs. Intriguingly, mycobacteria increased SUMOylation of many of the above molecules. Furthermore, inhibiting SUMOylation in DCs primed T cells that in turn attenuated bacterial burden in infected macrophages. These findings demonstrate that SUMOylation pathway is exploited by mycobacteria to thwart protective host immune responses.

Cap2-HAP complex is a critical transcriptional regulator that has dual but contrasting roles in regulation of iron homeostasis in Candida albicans.

Iron homeostasis is highly regulated in organisms across evolutionary time scale as iron is essential for various cellular processes. In a computational screen, we identified the Yap/bZIP domain family in Candida clade genomes. Cap2/Hap43 is essential for C. albicans growth under iron-deprivation conditions and for virulence in mouse. Cap2 has an amino-terminal bipartite domain comprising a fungal-specific Hap4-like domain and a bZIP domain. Our mutational analyses showed that both the bZIP and Hap4-like domains perform critical and independent functions for growth under iron-deprivation conditions. Transcriptome analysis conducted under iron-deprivation conditions identified about 16% of the C. albicans ORFs that were differentially regulated in a Cap2-dependent manner. Microarray data also suggested that Cap2 is required to mobilize iron through multiple mechanisms; chiefly by activation of genes in three iron uptake pathways and repression of iron utilizing and iron storage genes. The expression of HAP2, HAP32, and HAP5, core components of the HAP regulatory complex was induced in a Cap2-dependent manner indicating a feed-forward loop. In a feed-back loop, Cap2 repressed the expression of Sfu1, a negative regulator of iron uptake genes. Cap2 was coimmunoprecipitated with Hap5 from cell extracts prepared from iron-deprivation conditions indicating an in vivo association. ChIP assays demonstrated Hap32-dependent recruitment of Hap5 to the promoters of FRP1 (Cap2-induced) and ACO1 (Cap2-repressed). Together our data indicates that the Cap2-HAP complex functions both as a positive and a negative regulator to maintain iron homeostasis in C. albicans.

Suppression of TLR2-induced IL-12, reactive oxygen species, and inducible nitric oxide synthase expression by Mycobacterium tuberculosis antigens expressed inside macrophages during the course of infection.

We report the enrichment of and immune responses mediated by genes expressed by Mycobacterium tuberculosis inside macrophages as a function of time. Results indicate that M. tuberculosis expresses different genes at different times postinfection. Genes expressed early (day 1) following infection enhance M. tuberculosis-mediated activation of dendritic cells (DCs), whereas genes expressed later (day 5) in the infection prevent DC activation. However, all genes downmodulated MHC class I and II expression on infected macrophages, thus compromising their ability to interact with Ag-specific T cells. Day-1 and -5 genes downmodulated proinflammatory cytokine production from DCs, thus impairing signal 3 during DC-T cell cognate interactions. Consequently, T cells activated by Ag-experienced DCs secreted low levels of IFN-gamma and IL-17 but maintained high IL-10 secretion, thus inducing suppressor responses. Further characterization revealed that day-1 and -5 genes increased TLR2-induced expression of suppressors of cytokine signaling 1 from DCs and downmodulated IL-12 expression. In addition, day-1 and -5 genes prevented the generation of reactive oxygen species in DCs. In contrast, although day-5 genes increased TLR2-mediated suppressors of cytokine signaling 1 expression in macrophages, day-1 genes downmodulated the expression of inducible NO synthase 2. Similar downregulation of immune responses was observed upon exogenous stimulation with day-1 or -5 Ags. Finally, day-1 and -5 genes promoted enhanced survival of M. tuberculosis inside DCs and macrophages. These results indicate that M. tuberculosis genes, expressed inside infected macrophages as a function of time, collectively suppress protective immune responses by using multiple and complementary mechanisms.

N-acetylglucosamine kinase, Hxk1 is a multifaceted metabolic enzyme in model pathogenic yeast Candida albicans.

The sensing of environmental conditions such as nutrient availability and the ability to adapt and respond to changing conditions are crucial for the survival of living organisms. Evidence from several organisms have revealed that some metabolic enzymes act as sensors of nutrient status and regulate the expression of sets of genes required for nutrients utilization and condition specific environmental adaptation. Thus metabolic enzymes regulate the signaling pathway by acting as transcriptional regulators and providing required metabolites. The commensal yeast, Candida albicans has recently emerged as a model system for understanding the N-acetylglucosamine (GlcNAc) signaling pathway in eukaryotes. GlcNAc kinase (Hxk1), the first enzyme of the catabolic cascade, has been shown to perform several functions such as regulation of gene expression and regulation of the metabolic status of the cell thereby resulting in a change in cell morphology (yeast-hyphal transition, white-opaque switching), metabolic gene expression, synthesis of metabolic precursors, induction of glycolytic flux rate and biofilm formation. Here, in this review we have discussed various roles of Hxk1that have not been reported in other organisms previously. The enzyme exhibits dynamic changes in subcellular localization consistent with its expanded functions inside the cell. Thus Hxk1 in C. albicans orchestrates several dynamic cellular processes and this signaling system can act as a paradigm to understand the cell fate and metabolic specialization in other eukaryotes too. Still, the molecular cues involved in Hxk1 mediating functions are yet to be unveiled; the relationship between Hxk1 sensing and its signaling effects is also not understood yet.

Suppressors of cytokine signaling inhibit effector T cell responses during Mycobacterium tuberculosis infection.

Protective immune responses during Mycobacterium tuberculosis (M. tuberculosis) infection are regulated at multiple levels and critically dependent on the balance in the secretion of pro-inflammatory and regulatory cytokines. A key factor that governs this balance at the cellular level is suppressors of cytokine signaling (SOCS). We recently demonstrated that toll-like receptor 2 and dendritic cell (DC)-SIGNR1 differentially regulate SOCS1 expression in DCs during M. tuberculosis infection. This consecutively regulated IL-12 production and determined M. tuberculosis survival. In this study, we characterized the role of SOCS1 in regulating effector responses from CD4(+) and CD8(+) T cells during M. tuberculosis infection. Our data indicate that T cells from M. tuberculosis-infected mice show increased and differential association of SOCS1 with CD3 and CD28, when compared with uninfected mice. While SOCS1 displays increased association with CD3 than CD28 in CD4(+) T cells; SOCS1 is associated more with CD28 than CD3 in CD8(+) T cells. Further, SOCS1 shows increased association with IL-12 and IL-2 receptors in both CD4(+) and CD8(+) T cells from infected mice when compared with naive mice. Silencing SOCS1 in T cells increased signal transduction from T cell receptor (TCR) and CD28 with enhanced activation of key signaling molecules and proliferation. Significantly, SOCS1-silenced T cells mediated enhanced clearance of M. tuberculosis inside macrophages. Finally, adoptive transfer of SOCS1-silenced T cells in M. tuberculosis-infected mice mediated significant reduction in M. tuberculosis loads in spleen. These results exemplify the negative role played by SOCS1 during T cell priming and effector functions during M. tuberculosis infection.

Proteome analysis of Plasmodium falciparum extracellular secretory antigens at asexual blood stages reveals a cohort of proteins with possible roles in immune modulation and signaling.

The highly co-evolved relationship of parasites and their hosts appears to include modulation of host immune signals, although the molecular mechanisms involved in the host-parasite interplay remain poorly understood. Characterization of these key genes and their cognate proteins related to the host-parasite interplay should lead to a better understanding of this intriguing biological phenomenon. The malaria agent Plasmodium falciparum is predicted to export a cohort of several hundred proteins to remodel the host erythrocyte. However, proteins actively exported by the asexual intracellular parasite beyond the host red blood cell membrane (before merozoite egress) have been poorly investigated so far. Here we used two complementary methodologies, two-dimensional gel electrophoresis/MS and LC-MS/MS, to examine the extracellular secreted antigens at asexual blood stages of P. falciparum. We identified 27 novel antigens exported by P. falciparum in the culture medium of which some showed clustering with highly polymorphic genes on chromosomes, suggesting that they may encode putative antigenic determinants of the parasite. Immunolocalization of four novel secreted proteins confirmed their export beyond the infected red blood cell membrane. Of these, preliminary functional characterization of two novel (Sel1 repeat-containing) parasite proteins, PfSEL1 and PfSEL2 revealed that they down-regulate expression of cell surface Notch signaling molecules in host cells. Also a novel protein kinase (PfEK) and a novel protein phosphatase (PfEP) were found to, respectively, phosphorylate/dephosphorylate parasite-specific proteins in the extracellular culture supernatant. Our study thus sheds new light on malaria parasite extracellular secreted antigens of which some may be essential for parasite development and could constitute promising new drug targets.