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BACKGROUND: Bacille Calmette-Guérin (BCG) vaccine is protective against Tuberculosis (TB) in children, but its efficacy wanes with age. Consequently, determining if BCG revaccination augments anti-TB immunity in young adults in TB endemic regions is vital. METHODS: Two hundred healthy adults, BCG vaccinated at birth, were tested for their IFN-γ release assay (IGRA) status. Of these, 28 IGRA(+) and 30 IGRA(–) were BCG revaccinated, and 24 IGRA(+) and 23 IGRA(–) subjects served as unvaccinated controls. T and innate cell responses to mycobacterial antigens were analyzed by 14-color flow cytometry over 34 weeks. RESULTS: IFN-γ and/or IL-2 Ag85A- and BCG-specific CD4(+) and CD8(+) T cell responses were boosted by revacciantion at 4 and 34 weeks, respectively, and were > 2-fold higher in IGRA(+) compared with IGRA(–) vaccinees. Polyfunctional Ag85A, BCG, and mycobacterium tuberculosis (Mtb) latency Ag–specific (LTAg-specific) CD4(+) T cells expressing up to 8 cytokines were also significantly enhanced in both IGRA(+) and IGRA(–) vaccinees relative to unvaccinated controls, most markedly in IGRA(+) vaccinees. A focused analysis of Th17 responses revealed expansion of Ag85A-, BCG-, and LTAg-specific total IL-17A(+),IL-17F(+),IL-22(+), and IL-10(+) CD4(+) T cell effectors in both IGRA(+) and IGRA(–) subjects. Also, innate IFN-γ(+) NK/γδ/NKT cell responses were higher in both IGRA(+) and IGRA(–) vaccinees compared with controls. This is the first evidence to our knowledge that BCG revaccination significantly boosts antimycobacterial Th1/Th17 responses in IGRA(+) and IGRA(–) subjects. CONCLUSION: These data show that BCG revaccination is immunogenic in IGRA(–) and IGRA(+) subjects, implying that Mtb preinfection in IGRA(+) subjects does not impact immunogenicity. This has implications for public health and vaccine development strategies. FUNDING: This work was funded principally by DBT-NIH (BT/MB/Indo-US/HIPC/2013).

Induction of vast transcriptional programs is a central event of innate host responses to viral infections. Here we report a transcriptional program with potent antiviral activity, driven by E74-like ETS transcription factor 1 (ELF1). Using microscopy to quantify viral infection over time, we found that ELF1 inhibits eight diverse RNA and DNA viruses after multi-cycle replication. Elf1 deficiency results in enhanced susceptibility to influenza A virus infections in mice. ELF1 does not feed-forward to induce interferons, and ELF1’s antiviral effect is not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNA-seq revealed that the ELF1 transcriptional program is distinct from interferon signatures. Thus, ELF1 provides an additional layer of the innate host response, independent from the action of type I interferons., Author summary After decades of research on the innate immune system, we still struggle to understand exactly how this first line of defense protects cells against viral infections. Our gap in knowledge stems, on one hand, from the sheer number of effector genes, few of which have been characterized in mechanistic detail. On the other hand, our understanding of innate gene transcription is constantly evolving. We know that different regulatory mechanisms greatly influence the quality, magnitude, and timing of gene expression, all of which may contribute to the antiviral power of the innate response. Deciphering these regulatory mechanisms is indispensable for harnessing the power of innate immunity in novel antiviral therapies. Here, we report a novel transcriptional program as part of the cell-intrinsic immune system, raised by E74-like ETS transcription factor 1 (ELF1). ELF1 potently restricts multi-cycle propagation of all viruses tested in our study. Reduced levels of ELF1 significantly diminish host defenses against influenza A virus in vitro and in vivo, suggesting a critical but previously overlooked role of this ETS transcription factor. The ELF1 program is complex and comprises over 300 potentially antiviral genes, which are almost entirely distinct from those known to be induced by interferon. Taken together, our data provide evidence for a program of antiviral protection that expands the previously known arsenal of the innate immune response.

Coxsackievirus B3 is an enterovirus, with positive-sense single-stranded RNA genome containing `Internal Ribosome Entry Site' (IRES) in the 5MODIFIER LETTER PRIMEUTR. Once sufficient viral proteins are synthesized in the cell from the input RNA, viral template switches from translation to replication to synthesize negative-strand RNA. Inhibition of translation is a key step in regulating this switch as the positive-strand RNA template should be free of ribosomes to enable polymerase movement. In this study, we show how a host protein hnRNP C1/C2 inhibits viral RNA translation. hnRNP C1/C2 interacts with stem-loop V in the IRES and displaces poly-pyrimidine tract binding protein, a positive regulator of translation. We further demonstrate that hnRNP C1/C2 induces translation to replication switch, independently from the already known role of the ternary complex (PCBP2-3CD-cloverleaf RNA). These results suggest a novel function of hnRNP C1/C2 in template switching of positive-strand from translation to replication by a new mechanism. Using mathematical modelling, we show that the differential affinity of hnRNP C1/C2 for positive and negative-strand RNAs guides the final +/- RNA ratio, providing first insight in the regulation of the positive to negative-strand RNA ratio in enteroviruses.

During enteroviral infections, the canonical translation factor eukaryotic translation initiation factor 4 ? I (eIF4GI) is cleaved by viral protease 2A. The resulting C-terminal fragment is recruited by the viral internal ribosome entry site (IRES) for efficient translation of the viral RNA. However, the 2A protease is not present in the viral capsid and is synthesized only after the initial round of translation. This presents the conundrum of how the initial round of translation occurs in the absence of the C-terminal eIF4GI fragment. Interestingly, the host protein DAP5 (also known as p97, eIF4GIII, and eIF4G2), an isoform of eIF4GI, closely resembles the eIF4GI C-terminal fragment produced after 2A protease?mediated cleavage. Using the Coxsackievirus B3 (CVB3) IRES as a model system, here we demonstrate that DAP5, but not the full-length eIF4GI, is required for CVB3 IRES activity for translation of input viral RNA. Additionally, we show that DAP5 is specifically required by type I IRES but not by type II or type III IRES, in which cleavage of eIF4GI has not been observed. We observed that both DAP5 and C-terminal eIF4GI interact with CVB3 IRES in the same region, but DAP5 exhibits a lower affinity for CVB3 IRES compared with the C-terminal eIF4GI fragment. It appears that DAP5 is required for the initial round of viral RNA translation by sustaining a basal level of CVB3 IRES activity. This activity leads to expression of 2A protease and consequent robust CVB3 IRES-mediated translation by the C-terminal eIF4GI fragment.

Bacterial cold shock proteins (CSPs) function as RNA chaperones. To assess CSP's roles in the intracellular human pathogen Salmonella Typhimurium, we analyzed their expression in varied stress conditions. We found that cold shock protein E (cspE or STM14_0732) is up-regulated during bile salt-induced stress and that an S. Typhimurium strain lacking cspE (ΔcspE) displays dose-dependent sensitivity to bile salts, specifically to deoxycholate. We also found that an uncharacterized gene, yciF (STM14_2092), is up-regulated in response to bile stress in WT but not in the ΔcspE strain. Complementation with WT CspE, but not with a F30V CspE variant, abrogated the bile sensitivity of ΔcspE as did multicopy overexpression of yciF. Northern blotting experiments with rifampicin disclosed that the regulation of yciF expression is, most likely, due to the RNA-stabilizing activity of CspE. Importantly, electrophoretic mobility shift assays indicated that purified CspE, but not the F30V variant, directly binds yciF mRNA. We also observed that the extra-cytoplasmic stress-response (ESR) pathway is augmented in the bile-treated ΔcspE strain, as judged by induction of RpoE regulon genes (rpoE, degP, and rybB) and downstream ESR genes (hfq, rne, and PNPase). Moreover, the transcript levels of the porin genes, ompD, ompF, and ompC, were higher in bile salts-stressed ΔcspE and correlated with higher intracellular accumulation of the fluorescent DNA stain bisBenzimide H 33258, indicating greater cell permeability. In conclusion, our study has identified YciF, a CspE target involved in the regulation of porins and in countering bile stress in S. Typhimurium.

The development of immunocompetent T cells entails a complex pathway of differentiation in the thymus. Thymic atrophy occurs with ageing and during conditions such as malnutrition, infections and cancer chemotherapy. The comparative changes in thymic subsets under different modes of thymic atrophy and the mechanisms involved are not well characterized. These aspects were investigated, using mice infected with Salmonella Typhimurium, injection with lipopolysaccharide (LPS), an inflammatory but non‐infectious stimulus, etoposide (Eto), a drug used to treat some cancers, and dexamethasone (Dex), a steroid used in some inflammatory diseases. The effects on the major subpopulations of thymocytes based on multicolour flow cytometry studies were, first, the CD4(−) CD8(−) double‐negative (DN) cells, mainly DN2–4, were reduced with infection, LPS and Eto treatment, but not with Dex. Second, the CD8(+) CD3(lo) immature single‐positive cells (ISPs) were highly sensitive to infection, LPS and Eto, but not Dex. Third, treatment with LPS, Eto and Dex reduced all three subpopulations of CD4(+) CD8(+) double‐positive (DP) thymocytes, i.e. DP1, DP2 and DP3, but the DP3 subset was relatively more resistant during infection. Fourth, both CD4(+) and CD8(+) single‐positive (SP) thymocytes were lowered by Eto and Dex, but not during infection. Notably, LPS lowered CD4(+) SP subsets, whereas the CD8(+) SP subsets were relatively more resistant. Interestingly, the reactive oxygen species quencher, N‐acetyl cysteine, greatly improved the survival of thymocytes, especially DNs, ISPs and DPs, during infection and LPS treatment. The implications of these observations for the development of potential thymopoietic drugs are discussed.

The global mechanisms and associated molecular alterations that occur in drug-resistant mycobacteria are poorly understood. To address this, we obtain genomics data and then construct a genome-scale response network in isoniazid-resistant Mycobacterium smegmatis and apply a network-mining algorithm. Through this, we decipher global alterations in an unbiased manner and identify emergent vulnerabilities in resistant bacilli, of which redox response was prominent. Using phenotypic profiling, we find that resistant bacilli exhibit collateral sensitivity to several compounds that block antioxidant responses. We find that nanogram/milliliter concentrations of ebselen, vancomycin, and phenylarsine oxide, in combination with isoniazid, are highly effective against Mycobacterium tuberculosis H37Rv and three clinical drug-resistant strains. Dynamic measurements of cytoplasmic redox potential revealed a surprisingly diminished capacity of clinical drug-resistant strains to counteract oxidative stress, providing a mechanistic basis for efficient and synergistic mycobactericidal activity of the drug combinations. Ebselen and vancomycin appear to be promising repurposable drugs.

Vaccines that confer protection through induction of adaptive T-cell immunity rely on understanding T-cell epitope (TCE) evolution induced by immune escape. This is poorly understood in tuberculosis (TB), an ancient, chronic disease, where CD4 T-cell immunity is of recognized importance. We probed 905 functionally validated, curated human CD4 T cell epitopes in 79 Mycobacterium tuberculosis (Mtb) whole genomes from India. This screen resulted in identifying 64 mutated epitopes in these strains initially using a computational pipeline and subsequently verified by single nucleotide polymorphism (SNP) analysis. SNP based phylogeny revealed the 79 Mtb strains to cluster to East African Indian (EAI), Central Asian Strain (CAS), and Beijing (BEI) lineages. Eighty-nine percent of the mutated T-cell epitopes (mTCEs) identified in the 79 Mtb strains from India has not previously been reported. These mTCEs were encoded by genes with high nucleotide diversity scores including seven mTCEs encoded by six antigens in the top 10% of rapidly divergent Mtb genes encoded by these strains. Using a T cell functional assay readout, we demonstrate 62% of mTCEs tested to significantly alter CD4 T-cell IFN gamma and/or IL2 secretion with associated changes in predicted HLA-DR binding affinity: the gain of function mutations displayed higher predicted HLA-DR binding affinity and conversely mutations resulting in loss of function displayed lower predicted HLA-DR binding affinity. Most mutated antigens belonged to the cell wall/cell processes, and, intermediary metabolism and respiration families though all known Mtb proteins encoded mutations. Analysis of the mTCEs in an SNP database of 5,310 global Mtb strains identified 82% mTCEs to be significantly more prevalent in Mtb strains isolated from India, including 36 mTCEs identified exclusively in strains from India. These epitopes had a significantly higher predicted binding affinity to HLA-DR alleles that were highly prevalent in India compared to HLA-DR alleles rare in India, highlighting HLA-DR maybe an important driver of these mutations. This first evidence of region-specific TCE mutations potentially employed by Mtb to escape host immunity has important implications for TB vaccine design.

The factors contributing to antibiotic resistance in bacteria are an important area of study. Sodium salicylate (NaSal), a non-steroidal anti-inflammatory drug (NSAID), increases antibiotic resistance by inducing the expression of MarA, a transcription factor, which increases the AcrAB-TolC efflux pump. MarA is a substrate of Lon protease and the Delta lon strain displays a high degree of antibiotic resistance. This study was initiated to identify commonly used NSAIDs that may induce antibiotic resistance and to compare their efficacies with NaSal and acetyl salicylic acid (ASA). Quantitative real-time expression analysis revealed induction of marA and acrB by NaSal, ASA, acetaminophen (APAP) and ibuprofen. Further, dose studies demonstrated that NaSal and ASA induce resistance at similar to 2 mM while APAP and ibuprofen induce resistance at similar to 5-10 mM. To dissect the roles of key molecules, atomic force microscopy and functional studies were performed using WT, Delta lon, Delta marA, Delta acrB, Delta lon Delta marA and Delta lon Delta acrB strains. The induction of antibiotic resistance by NaSal, ASA and APAP is relatively higher and is partly dependent on marA, whereas ibuprofen which induces lower antibiotic resistance shows complete marA dependence. Notably, NaSal, ASA, APAP and ibuprofen induce antibiotic resistance in an acrB-dependent manner. The possible significance of some NSAIDs in inducing antibiotic resistance is discussed.

Sepsis, a leading cause of death in intensive care units, is primarily caused due to an exaggerated immune response. The hyperactive inflammatory response mediated by immune cells against infectious organisms and their toxins results in host cell death and tissue damage, the hallmarks of septic shock. Therefore, molecules that modulate inflammatory responses are attractive therapeutic targets for sepsis. Nitric oxide (NO) is a signaling molecule, which is implicated in regulating diverse immune functions. Although, the protective roles of NO in infectious diseases are well documented, its importance in sepsis is controversial. In the present study, the effects of intra-peritoneal injection of mice with Salmonella Typhimurium, a Gram-negative intracellular pathogen, were studied which leads to a rapid upregulation of serum cytokines and infiltration of neutrophils to the peritoneal cavity. Surprisingly, the induction of inflammatory cytokines and chemokines, e.g. IL6 and CCL2, and the infiltration of neutrophils into the peritoneal cavity are mitigated in mice lacking Nitric oxide synthase 2 (NOS2). The reduced inflammatory response in Nos2(-/-) mice is accompanied by greater bacterial burden in the peritoneal cavity, lower thymic atrophy, higher liver damage and cardiovascular dysfunction followed by decreased survival. However, no significant differences are observed in other responses between C57BL/6 wild type (WT) and Nos2(-/-) mice: induction of glucocorticoids, phagocytic ability and apoptosis of peritoneal cells. This study clearly highlights the NOS2-dependent and - independent responses in this mouse model of peritonitis induced sepsis. Importantly, pre-treatment of Nos2(-/-) mice with DETA-NO, a NO donor, upon infection, restores neutrophil recruitment, reduces bacterial numbers in the peritoneal cavity, improves liver and cardio-vascular function and enhances survival. Interestingly, DETA-NO treatment does not significantly increase the survival of infected WT mice. The implications of these results and the complex roles of NO as a target molecule during sepsis are discussed.

Mycobacteria propelled modulation of host responses is of considerable interest in the face of emerging drug resistance. Although it is known that Abl tyrosine kinases affect entry and persistence of mycobacteria, mechanisms that couple c-Abl to proximal signaling pathways during immunity are poorly understood. Loss-of-function of c-Abl through Imatinib, in a mouse model of tuberculosis or RNA interference, identified bone morphogenesis protein (BMP) signaling as its cellular target. We demonstrate that c-Abl promotes mycobacterial survival through epigenetic modification brought about by KAT5-TWIST1 at Bmp loci. c-Abl-BMP signaling deregulated iNOS, aggravating the inflammatory balance. Interestingly, BMP signaling was observed to have far-reaching effects on host immunity, as it attenuated TLR3 pathway by engaging miR27a. Significantly, these events were largely mediated via WhiB3 and DosR/S/T but not SecA signaling pathway of mycobacteria. Our findings suggest molecular mechanisms of host pathways hijacked by mycobacteria and expand our understanding of c-Abl inhibitors in potentiating innate immune responses.

Bactericidal permeability increasing protein (BPI), a 55–60 kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

The proteins of Plasmodium, the malaria parasite, are strikingly rich in asparagine. Plasmodium depends primarily on host haemoglobin degradation for amino acids and has a rudimentary pathway for amino acid biosynthesis, but retains a gene encoding asparagine synthetase (AS). Here we show that deletion of AS in Plasmodium berghei (Pb) delays the asexual- and liver-stage development with substantial reduction in the formation of ookinetes, oocysts and sporozoites in mosquitoes. In the absence of asparagine synthesis, extracellular asparagine supports suboptimal survival of PbAS knockout (KO) parasites. Depletion of blood asparagine levels by treating PbASKO-infected mice with asparaginase completely prevents the development of liver stages, exflagellation of male gametocytes and the subsequent formation of sexual stages. In vivo supplementation of asparagine in mice restores the exflagellation of PbASKO parasites. Thus, the parasite life cycle has an absolute requirement for asparagine, which we propose could be targeted to prevent malaria transmission and liver infections., Malaria parasites obtain amino acids primarily from the host, but possess a gene encoding a putative asparagine synthetase. Here, the authors show that this enzyme is functional and that asparagine is crucial for the development of the parasite's sexual stages in mosquitoes and liver stages in mice.

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N-5, N-10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in alpha-lcetoacid dehydrogenase reactions. (C) 2014 Elsevier B.V. All rights reserved.