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We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apoptosis by upregulating the expression of Bcl2 and Inhibitor of Apoptosis (IAP). In addition, recent reports from our lab showed that stimulation of either macrophages or BMDCs results in the significant upregulation of Bcl2. In this report, we delineate the role of Bcl2 in mediating defense responses from dendritic cells (BMDCs) during mycobacterial infection. Inhibiting Bcl2 led to a significant decrease in intracellular bacterial burden in BMDCs. To further characterize the role of Bcl2 in modulating defense responses, we inhibited Bcl2 in BMDCs as well as human PBMCs to monitor their activation and functional status in response to mycobacterial infection and stimulation with M. tb antigen Rv3416. Inhibiting Bcl2 generated protective responses including increased expression of co-stimulatory molecules, oxidative burst, pro-inflammatory cytokine expression and autophagy. Finally, co-culturing human PBMCs and BMDCs with antigen-primed T cells increased their proliferation, activation and effector function. These results point towards a critical role for Bcl2 in regulating BMDCs defense responses to mycobacterial infection.

Staphylococcus aureus (S. aureus) is a gram-positive bacteria, which causes various fatal respiratory infections including pneumonia. The emergence of Methicillin-Resistance Staphylococcus aureus (MRSA) demands a thorough understanding of host-pathogen interactions. Here we report the role of calcium in regulating defence responses of S. aureus in macrophages. Regulating calcium fluxes in cells by different routes differentially governs the expression of T cell costimulatory molecule CD80 and Th1 promoting IL-12 receptor. Inhibiting calcium influx from extracellular medium increased expression of IFN-γ and IL-10 while blocking calcium release from the intracellular stores inhibited TGF-β levels. Blocking voltage-gated calcium channels (VGCC) inhibited the expression of multiple cytokines. While VGCC regulated the expression of apoptosis protein Bax, extracellular calcium-regulated the expression of Cytochrome-C. Similarly, VGCC regulated the expression of autophagy initiator Beclin-1. Blocking VGCC or calcium release from intracellular stores promoted phagosome-lysosome fusion, while activating VGCC inhibited phagosomelysosome fusion. Finally, calcium homeostasis regulated intracellular growth of Staphylococcus, although using different mechanisms. While blocking extracellular calcium influx seems to rely on IFN-γ and IL-12Rβ receptor mediated reduction in bacterial survival, blocking either intracellular calcium release or via VGCC route seem to rely on enhanced autophagy mediated reduction of intracellular bacterial survival. These results point to fine-tuning of defence responses by routes of calcium homeostasis.

Interferon-gamma (IFN-γ) is a key cytokine that mediates immunity to tuberculosis (TB). Mycobacterium tuberculosis (M. tb) is known to downregulate the surface expression of IFN-γ receptor (IFN-γR) on macrophages and peripheral blood mononuclear cells (PBMCs) of patients with active TB disease. Many M. tb antigens also downmodulate IFN-γR levels in macrophages when compared with healthy controls. In the current study, we aimed at deciphering key factors involved in M. tb mediated downregulation of IFN-γR levels on macrophage surface. Our data showed that both M. tb H37Rv and M. bovis BCG infections mediate downmodulation of IFN-γR on human macrophages. This downmodulation is regulated at the level of TLR signaling pathway, second messengers such as calcium and cellular kinases i.e. PKC and ERK-MAPK, indicating that fine tuning of calcium response is critical to maintaining IFN-γR levels on macrophage surface. In addition, genes in the calcium and cysteine protease pathways which were previously identified by us to play a negative role during M. tb infection, also regulated IFN-γR expression. Thus, modulations in IFN-γR levels by utilizing host machinery may be a key immune suppressive strategy adopted by the TB pathogen to ensure its persistence and thwart host defense.

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.

We previously reported that

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.

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.

TAF6, bearing the histone H4-like histone-fold domain (HFD), is a subunit of the core TAF module in TFIID and SAGA transcriptional regulatory complexes. We isolated and characterized several yeast TAF6 mutants bearing amino acid substitutions in the HFD, the middle region or the HEAT repeat domain. The TAF6 mutants were highly defective for transcriptional activation by the Gcn4 and Gal4 activators. CHIP assays showed that the TAF6-HFD and the TAF6-HEAT domain mutations independently abrogated the promoter occupancy of TFIID and SAGA complex in vivo. We employed genetic and biochemical assays to identify the relative contributions of the TAF6 HFD and HEAT domains. First, the temperature-sensitive phenotype of the HEAT domain mutant was suppressed by overexpression of the core TAF subunits TAF9 and TAF12, as well as TBP. The HFD mutant defect, however, was suppressed by TAF5 but not by TAF9, TAF12 or TBP. Second, the HEAT mutant but not the HFD mutant was defective for growth in the presence of transcription elongation inhibitors. Third, coimmunoprecipitation assays using yeast cell extracts indicated that the specific TAF6 HEAT domain residues are critical for the interaction of core TAF subunits with the SAGA complex but not with TFIID. The specific HFD residues in TAF6, although required for heterodimerization between TAF6 and TAF9 recombinant proteins, were dispensable for association of the core TAF subunits with TFIID and SAGA in yeast cell extracts. Taken together, the results of our studies have uncovered the non-overlapping requirement of the evolutionarily conserved HEAT domain and the HFD in TAF6 for transcriptional activation.

Microorganisms are known to devise various strategies to thwart protective responses by the host. One such strategy is to incorporate sequences and domains in their genes/proteins that have similarity to various domains of the host proteins. In this study, we report that Mycobacterium tuberculosis protein Rv3529c exhibits significant similarity to the death domain of the TLR pathway adaptor protein MyD88. Incubation of macrophages with Rv3529c specifically inhibited TLR2-mediated proinflammatory responses. This included attenuated oxidative burst, reduced phosphorylation of MAPK-ERK, reduced activation of transcription factor NF-κB and reduced secretion of proinflammatory cytokines IFN-γ, IL-6, and IL-17A with a concomitant increased secretion of suppressor cytokines IL-10 and TGF-β. Importantly, Rv3529c significantly inhibited TLR2-induced association of MyD88 with IRAK1 by competitively binding with IRAK1. Further, Rv3529c mediated inhibition of apoptosis and phagosome–lysosome fusion. Lastly, incubation of macrophages with Rv3529c increased bacterial burden inside macrophages. The data presented show another strategy evolved by M. tuberculosis toward immune evasion that centers on incorporating sequences in proteins that are similar to crucial proteins in the innate immune system of the host.

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.

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.

Pathogen-associated molecular patterns (PAMPs) associated with viral and bacterial infections trigger multiple inflammatory pathways which may result in oxidative stress driven toxicity, tissue fibrosis organ dysfunction and ageing. Inflammatory events need high energy demands and predominantly depends on the glycolysis. Thus, energy metabolism of the inflammatory events can be targeted to reducing the magnitude of the PAMPs driven inflammation and preventing tissue toxicity. Here we propose that 2-DG, a glycolytic inhibitor, and a potential Energy Restriction Mimetic agent (ERMA) can modulate inflammatory events and can prevent the development of acute as well as chronic pathology. For this study we induced LPS (bacterial PAMP) induced endotoxemia in mice which models infection associated inflammatory acute inflammatory events, tissue damage and organ dysfunction. 2-DG fed mice (0.4% w/v in drinking water) showed reduced LPS driven oxidative stress and capillary damage in lungs. Administration of 2-DG also reduced LPS induced spike in inflammatory cytokines (TNF, IL6 and IL1β) in the BALF and serum. Lungs of 2-DG fed mice showed lesser infiltration of inflammatory cells and reduced inflammatory signaling activation. 2-DG also downregulated the ex-vivo and in-vivo migration of the PMNCs. Furthermore, 2-DG also reduced the activation of the macrophage cells (RAW264.7) which was seen with reduction and the glycolysis and increased mitochondrial functions. Our data suggest that 2-DG administration as ERMA in drinking water can prevent pathogenic exposure driven inflammatory events which may prevent acute as well as chronic inflammatory disorders.

Obesity is a major risk factor for type 2 diabetes, NAFLD, chronic diseases and cancer. Insulin resistance, oxidative stress, high ectopic lipid levels and meta-inflammation are the mechanisms proposed to play a leading role in the morbidity associated with obesity. Energy restriction mimetics (ERMAs) has also been shown earlier to reduce the scale and the severity of these disorders by mimicking the physiological effects of the Energy Restriction. In present study we propose that the use of 2-DG as ERMA can be effective in regulating the High Fat Diet (HFD) induced obesity. Effect of 2-DG (0.4% w/v in drinking water) on the HFD and Insulin Resistance (IR). HFD induced change in body weight, adipose tissue mass, and ectopic lipid levels was assessed as the measure of obesity.IR and glucose levels were also estimated to evaluate the effect of 2-DG on the insulin sensitivity in HFD mice. 2-DG significantly altered HFD induced increase in the mice body weight, epididymal White Adipose Tissue (WAT) and liver weight. 2-DG fed mice also showed reduced lipid levels in serum and liver. Furthermore, 2-DG also reduced the oxidative damage in the liver with concomitant increase in enzymatic (SOD and Catalase) and non-enzymatic (reduced Glutathione) antioxidant levels. 2-DG fed mice also showed reduced levels of Leptin, IL-6 and TGF-β which are early drivers of the etiology of the metabolic diseases. Our results suggest that 2-DG as ERMA can prevent obesity and etiology of associated disorders. However, more relevant models are needed to further strengthen these observation

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.

Iron is an essential micronutrient for living cells. Candida albicans, the predominant human fungal pathogen, thrives under diverse environments with vastly different iron levels in the mammalian host. Therefore, to tightly control iron homeostasis, C. albicans has evolved a set of transcriptional regulators that cooperate to either upregulate or downregulate transcription of iron uptake genes or iron utilization genes. Cap2/Hap43, a critical transcriptional regulator, contains multiple conserved protein domains. In this study, we carried out mutational analyses to identify the functional roles of the conserved protein domains in Cap2. Our results show that the bZIP, HAP4L, and the C-terminal domain are each required for Cap2 transcriptional activity. Thus, Cap2 employs multiple, disparate protein domains for regulation of iron homeostasis in C. albicans., Iron is required for growth and metabolism by virtually all organisms. The human fungal pathogen Candida albicans has evolved multiple strategies to acquire iron. The Cap2/Hap43 transcriptional regulator, essential for robust virulence of C. albicans, controls iron homeostasis gene expression by promoter binding and repression of iron utilization genes. The expression of iron uptake genes is also dependent on Cap2, although Cap2 was not recruited to its promoters. Cap2, bearing the conserved bipartite HAP4L-bZIP domain, also contains multiple blocks of amino acids that form the highly conserved carboxyl-terminal region. In this study, we sought to identify the requirements of the different domains for Cap2 function. We constructed a series of mutants bearing either point mutations or deletions in the conserved domains and examined Cap2 activity. Deletion of the highly conserved extreme C-terminal region did not impair expression of Cap2 mutant protein but impaired cell growth and expression of iron homeostasis genes under iron-depleted conditions. Mutations in the amino-terminal HAP4L and basic leucine zipper (bZIP) domains also impaired growth and gene expression. Furthermore, chromatin immunoprecipitation (ChIP) assays showed that the HAP4L domain and the bZIP domain are both essential for Cap2 recruitment to ACO1 and CYC1 promoters. Unexpectedly, the C-terminal conserved region was also essential for Cap2 promoter recruitment. Thus, our results suggest that Cap2 employs multiple evolutionarily conserved domains, including the C-terminal domain for its transcriptional activity. IMPORTANCE Iron is an essential micronutrient for living cells. Candida albicans, the predominant human fungal pathogen, thrives under diverse environments with vastly different iron levels in the mammalian host. Therefore, to tightly control iron homeostasis, C. albicans has evolved a set of transcriptional regulators that cooperate to either upregulate or downregulate transcription of iron uptake genes or iron utilization genes. Cap2/Hap43, a critical transcriptional regulator, contains multiple conserved protein domains. In this study, we carried out mutational analyses to identify the functional roles of the conserved protein domains in Cap2. Our results show that the bZIP, HAP4L, and the C-terminal domain are each required for Cap2 transcriptional activity. Thus, Cap2 employs multiple, disparate protein domains for regulation of iron homeostasis in C. albicans.

Complement system is the first line of human defence against intruding pathogens and is recognized as a potentially useful therapeutic target. Human malaria parasite

Multiple strategies evolved by Mycobacterium tuberculosis (M. tb) have contributed to its successful prevalence. We previously identified specific genes in the cysteine protease and calcium-calmodulin pathways that regulated immune responses from dendritic cells (DCs). In this study we have characterized the role of neddylation in regulating various defense responses from DCs during mycobacterial infection. Neddylation is a process that is similar to ubiquitination. It however has its own enzyme machinery. It is coupled to ubiquitination and is important for maintaining cellular homeostasis. Here we show that stimulation of DCs with M. tb antigens Rv2463 and Rv3416 as well as infection with live M. tb modulates the expression levels of key proteins in the neddylation pathway. Further, stimulation with the two antigens promoted the association of NEDD8 with its target Cullin-1. The modulation in the expression levels of NEDD8 and SENtrin specific Protein 8 (SENP8) by the two antigens was in a calcium, MAPK and TLR dependent mechanism. Further, knockdown of specific genes of neddylation promoted the generation of oxidative burst, promoted phagolysosome fusion in mycobacteria infected DCs and induced higher expression of autophagy and apoptosis associated proteins in DCs. These results point toward a unique strategy employed by mycobacteria and its antigens towards immune suppression via modulating neddylation in DCs.

A common function of the TFIID and SAGA complexes, which are recruited by transcriptional activators, is to deliver TBP to promoters to stimulate transcription. Neither the relative contributions of the five shared TBP-associated factor (TAF) subunits in TFIID and SAGA nor the requirement for different domains in shared TAFs for transcriptional activation is well understood. In this study, we uncovered the essential requirement for the highly conserved C-terminal region (CRD) of Taf9, a shared TAF, for transcriptional activation in yeast. Transcriptome profiling performed under Gcn4-activating conditions showed that the Taf9 CRD is required for induced expression of ∼9% of the yeast genome. The CRD was not essential for the Taf9-Taf6 interaction, TFIID or SAGA integrity, or Gcn4 interaction with SAGA in cell extracts. Microarray profiling of a SAGA mutant (spt20Δ) yielded a common set of genes induced by Spt20 and the Taf9 CRD. Chromatin immunoprecipitation (ChIP) assays showed that, although the Taf9 CRD mutation did not impair Gcn4 occupancy, the occupancies of TFIID, SAGA, and the preinitiation complex were severely impaired at several promoters. These results suggest a crucial role for the Taf9 CRD in genome-wide transcription and highlight the importance of conserved domains, other than histone fold domains, as a common determinant for TFIID and SAGA functions.

In vitiligo, chronic loss of melanocytes and consequent absence of melanin from the epidermis presents a challenge for long-term tissue maintenance. The stable vitiligo patches are known to attain an irreversible depigmented state. However, the molecular and cellular processes resulting in this remodeled tissue homeostasis is unclear. To investigate the complex interplay of inductive signals and cell intrinsic factors that support the new acquired state, we compared the matched lesional and non-lesional epidermis obtained from stable non-segmental vitiligo subjects. Hierarchical clustering of genome-wide expression of transcripts surprisingly segregated lesional and non-lesional samples in two distinct clades, despite the apparent heterogeneity in the lesions of different vitiligo subjects. Pathway enrichment showed the expected downregulation of melanogenic pathway and a significant downregulation of cornification and keratinocyte differentiation processes. These perturbations could indeed be recapitulated in the lesional epidermal tissue, including blunting of rete-ridges, thickening of stratum corneum and increase in the size of corneocytes. In addition, we identify marked increase in the putrescine levels due to the elevated expression of spermine/spermidine acetyl transferase. Our study provides insights into the intrinsic self-renewing ability of damaged lesional tissue to restore epidermal functionality in vitiligo.

Microorganisms are known to devise various strategies to thwart protective responses by the host. One such strategy is to incorporate sequences and domains in their genes/proteins that have similarity to various domains of the host proteins. In this study, we report that

The host nutritional environment is a key determinant for survival of human pathogens including C. albicans. Iron is an essential micronutrient for the host as well as for C. albicans. However, iron sequestration in host proteins confers ‘nutritional immunity’. In this chapter, we have reviewed our current understanding of the different iron uptake systems, the associated genes, and their transcriptional regulation in C. albicans. Besides, iron acquisition is essential for C. albicans virulence and iron homeostasis is also interlinked with lipid homeostasis and multidrug resistance. We have also provided an atlas of all annotated C. albicans iron acquisition genes with the attendant gene expression, and phenotype data including virulence.