Immunological consequences of intragenus conservation of Mycobacterium tuberculosis T-cell epitopes.

A previous unbiased genome-wide analysis of CD4 Mycobacterium tuberculosis (MTB) recognition using peripheral blood mononuclear cells from individuals with latent MTB infection (LTBI) or nonexposed healthy controls (HCs) revealed that certain MTB sequences were unexpectedly recognized by HCs. In the present study, it was found that, based on their pattern of reactivity, epitopes could be divided into LTBI-specific, mixed reactivity, and HC-specific categories. This pattern corresponded to sequence conservation in nontuberculous mycobacteria (NTMs), suggesting environmental exposure as an underlying cause of differential reactivity. LTBI-specific epitopes were found to be hyperconserved, as previously reported, whereas the opposite was true for NTM conserved epitopes, suggesting that intragenus conservation also influences host pathogen adaptation. The biological relevance of this observation was demonstrated further by several observations. First, the T cells elicited by MTB/NTM cross-reactive epitopes in HCs were found mainly in a CCR6(+)CXCR3(+) memory subset, similar to findings in LTBI individuals. Thus, both MTB and NTM appear to elicit a phenotypically similar T-cell response. Second, T cells reactive to MTB/NTM-conserved epitopes responded to naturally processed epitopes from MTB and NTMs, whereas T cells reactive to MTB-specific epitopes responded only to MTB. Third, cross-reactivity could be translated to antigen recognition. Several MTB candidate vaccine antigens were cross-reactive, but others were MTB-specific. Finally, NTM-specific epitopes that elicit T cells that recognize NTMs but not MTB were identified. These epitopes can be used to characterize T-cell responses to NTMs, eliminating the confounding factor of MTB cross-recognition and providing insights into vaccine design and evaluation.

Transcriptional profile of tuberculosis antigen-specific T cells reveals novel multifunctional features.

In latent tuberculosis infection (LTBI) spread of the bacteria is contained by a persistent immune response, which includes CD4(+) T cells as important contributors. In this study we show that TB-specific CD4(+) T cells have a characteristic chemokine expression signature (CCR6(+)CXCR3(+)CCR4(-)), and that the overall number of these cells is significantly increased in LTBI donors compared with healthy subjects. We have comprehensively characterized the transcriptional signature of CCR6(+)CXCR3(+)CCR4(-) cells and found significant differences to conventional Th1, Th17, and Th2 cells, but no major changes between healthy and LTBI donors. CCR6(+)CXCR3(+)CCR4(-) cells display lineage-specific signatures of both Th1 and Th17 cells, but also have a unique gene expression program, including genes associated with susceptibility to TB, enhanced T cell activation, enhanced cell survival, and induction of a cytotoxic program akin to CTL cells. Overall, the gene expression signature of CCR6(+)CXCR3(+)CCR4(-) cells reveals characteristics important for controlling latent TB infections.

The nutrient supplying capabilities of Uzinura, an endosymbiont of armoured scale insects.

An emerging common physiological feature of plant sap-feeding insects is the presence of bacterial endosymbionts capable of providing essential nutrients to their host. These microbial partners are inviable outside of specialized host tissues, and therefore a cultivation-independent approach, namely high-throughput next-generation genome sequencing, can be used to characterize their gene content and metabolic potential. To this end, we sequenced the first complete genome of the obligate endosymbiont, Candidatus 'Uzinura diaspidicola', of armoured scale insects. At 263 431 bp, Uzinura has an extremely reduced genome that is composed largely of genes encoding enzymes involved in translation and amino acid biosynthesis. The tiny size of the Uzinura genome parallels that observed in some other insect endosymbionts. Despite this extreme genome reduction, the absence of a known obligate partner bacterial symbiont suggests that Uzinura alone can supply sufficient nutrients to its host.

Genome sequence of Blattabacterium sp. strain BGIGA, endosymbiont of the Blaberus giganteus cockroach.

Cockroaches harbor the obligate flavobacterial endosymbiont Blattabacterium sp., which resides within the host's bacteriocytes and can recycle ammonia and urea nitrogenous wastes into amino acids for the host. We report the complete genome sequence of the Blattabacterium sp. associated with the giant roach Blaberus giganteus.

Genome shrinkage and loss of nutrient-providing potential in the obligate symbiont of the primitive termite Mastotermes darwiniensis.

Beneficial microbial associations with insects are common and are classified as either one or a few intracellular species that are vertically transmitted and reside intracellularly within specialized organs or as microbial assemblages in the gut. Cockroaches and termites maintain at least one if not both beneficial associations. Blattabacterium is a flavobacterial endosymbiont of nearly all cockroaches and the termite Mastotermes darwiniensis and can use nitrogenous wastes in essential amino acid and vitamin biosynthesis. Key changes during the evolutionary divergence of termites from cockroaches are loss of Blattabacterium, diet shift to wood, acquisition of a specialized hindgut microbiota, and establishment of advanced social behavior. Termite gut microbes collaborate to fix nitrogen, degrade lignocellulose, and produce nutrients, and the absence of Blattabacterium in nearly all termites suggests that its nutrient-provisioning role has been replaced by gut microbes. M. darwiniensis is a basal, extant termite that solely retains Blattabacterium, which would show evidence of relaxed selection if it is being supplanted by the gut microbiome. This termite-associated Blattabacterium genome is ∼8% smaller than cockroach-associated Blattabacterium genomes and lacks genes underlying vitamin and essential amino acid biosynthesis. Furthermore, the M. darwiniensis gut microbiome membership is more consistent between individuals and includes specialized termite gut-associated bacteria, unlike the more variable membership of cockroach gut microbiomes. The M. darwiniensis Blattabacterium genome may reflect relaxed selection for some of its encoded functions, and the loss of this endosymbiont in all remaining termite genera may result from its replacement by a functionally complementary gut microbiota.

Temporal regulation of Cat-1 (cationic amino acid transporter-1) gene transcription during endoplasmic reticulum stress.

Expression of the Cat-1 gene (cationic amino acid transporter-1) is induced in proliferating cells and in response to a variety of stress conditions. The expression of the gene is mediated via a TATA-less promoter. In the present study we show that an Sp1 (specificity protein 1)-binding site within a GC-rich region of the Cat-1 gene controls its basal expression and is important for induction of the gene during the UPR (unfolded protein response). We have shown previously that induction of Cat-1 gene expression during the UPR requires phosphorylation of the translation initiation factor eIF2alpha (eukaryotic initiation factor 2alpha) by PERK (protein-kinase-receptor-like endoplasmic reticulum kinase), one of the signalling pathways activated during the UPR. This leads to increased translation of the transcription factor ATF4 (activating transcription factor 4). We also show that a second signalling pathway is required for sustained transcriptional induction of the Cat-1 gene during the UPR, namely activation of IRE1 (inositol-requiring enzyme 1) leading to alternative splicing of the mRNA for the transcription factor XBP1 (X-box-binding protein 1). The resulting XBP1s (spliced XBP1) can bind to an ERSE (endoplasmic-reticulum-stress-response-element), ERSE-II-like, that was identified within the Cat-1 promoter. Surprisingly, eIF2alpha phosphorylation is required for accumulation of XBP1s. We propose that the signalling via phosphorylated eIF2alpha is required for maximum induction of Cat-1 transcription during the UPR by inducing the accumulation of both ATF4 and XBP1s.

A bifunctional intronic element regulates the expression of the arginine/lysine transporter Cat-1 via mechanisms involving the purine-rich element binding protein A (Pur alpha).

Expression of the arginine/lysine transporter Cat-1 is highly induced in proliferating and stressed cells via mechanisms that include transcriptional activation. A bifunctional INE (intronic element) within the first intron of the Cat-1 gene was identified and characterized in this study. The INE had high sequence homology to an amino acid response element and was shown to act as a transcriptional enhancer in unstressed cells by binding the transcription factor, purine-rich element binding protein A (Pur alpha). During endoplasmic reticulum stress, binding of Pur alpha to the INE decreased; the element acted as a positive regulator in early stress by binding of the transcription factor ATF4 and as a negative regulator in prolonged stress by binding the stress-induced C/EBP family member, CHOP. We conclude that transcriptional control of the Cat-1 gene is tightly controlled by multiple cis-DNA elements, contributing to regulation of cationic amino acid transport for cell growth and proliferation. In addition, we propose that genes may use stress-response elements such as the INE to support basal expression in the absence of stress.

hipBA toxin-antitoxin systems mediate persistence in Caulobacter crescentus.

Antibiotic persistence is a transient phenotypic state during which a bacterium can withstand otherwise lethal antibiotic exposure or environmental stresses. In Escherichia coli, persistence is promoted by the HipBA toxin-antitoxin system. The HipA toxin functions as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neutralizes the toxin. E. coli HipA inactivates the glutamyl-tRNA synthetase GltX, which inhibits translation and triggers the highly conserved stringent response. Although hipBA operons are widespread in bacterial genomes, it is unknown if this mechanism is conserved in other species. Here we describe the functions of three hipBA modules in the alpha-proteobacterium Caulobacter crescentus. The HipA toxins have different effects on growth and macromolecular syntheses, and they phosphorylate distinct substrates. HipA1 and HipA2 contribute to antibiotic persistence during stationary phase by phosphorylating the aminoacyl-tRNA synthetases GltX and TrpS. The stringent response regulator SpoT is required for HipA-mediated antibiotic persistence, but persister cells can form in the absence of all hipBA operons or spoT, indicating that multiple pathways lead to persister cell formation in C. crescentus.

A feedback transcriptional mechanism controls the level of the arginine/lysine transporter cat-1 during amino acid starvation.

The adaptive response to amino acid limitation in mammalian cells inhibits global protein synthesis and promotes the expression of proteins that protect cells from stress. The arginine/lysine transporter, cat-1, is induced during amino acid starvation by transcriptional and post-transcriptional mechanisms. It is shown in the present study that the transient induction of cat-1 transcription is regulated by the stress response pathway that involves phosphorylation of the translation initiation factor, eIF2 (eukaryotic initiation factor-2). This phosphorylation induces expression of the bZIP (basic leucine zipper protein) transcription factors C/EBP (CCAAT/enhancer-binding protein)-beta and ATF (activating transcription factor) 4, which in turn induces ATF3. Transfection experiments in control and mutant cells, and chromatin immunoprecipitations showed that ATF4 activates, whereas ATF3 represses cat-1 transcription, via an AARE (amino acid response element), TGATGAAAC, in the first exon of the cat-1 gene, which functions both in the endogenous and in a heterologous promoter. ATF4 and C/EBPbeta activated transcription when expressed in transfected cells and they bound as heterodimers to the AARE in vitro. The induction of transcription by ATF4 was inhibited by ATF3, which also bound to the AARE as a heterodimer with C/EBPbeta. These results suggest that the transient increase in cat-1 transcription is due to transcriptional activation caused by ATF4 followed by transcriptional repression by ATF3 via a feedback mechanism.

A novel feedback loop regulates the response to endoplasmic reticulum stress via the cooperation of cytoplasmic splicing and mRNA translation.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers transcriptional and translational reprogramming. This unfolded protein response (UPR) protects cells during transient stress and can lead to apoptosis during prolonged stress. Two key mediators of the UPR are PKR-like ER kinase (PERK), which phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in decreased protein synthesis, and the α subunit of inositol-requiring enzyme 1 (IRE1α), which initiates cytoplasmic splicing of the mRNA encoding the transcription factor X-box binding protein 1 (XBP1). XBP1 induces transcription of genes involved in protein quality control. This report describes cross talk between these two pathways: phosphorylation of eIF2α was required for maximal induction of spliced XBP1 (XBP1s) protein levels via a mechanism that involved stabilization of XBP1s mRNA. By using mouse embryo fibroblasts deficient in UPR signaling pathways, we demonstrate that stress-induced stabilization of XBP1s mRNA requires cytoplasmic splicing of the mRNA and inhibition of its translation. Because the XBP1s protein promotes transcription of its own gene, the UPR-induced mRNA stabilization is part of a positive feedback loop that induces XBP1s protein accumulation and transcription of target genes during stress. We propose a model in which eIF2α phosphorylation-mediated control of mRNA turnover is a molecular switch that regulates the stress response transcription program and the ER's capacity for protein folding during stress.

Molecular symbiosis of CHOP and C/EBP beta isoform LIP contributes to endoplasmic reticulum stress-induced apoptosis.

Induction of the transcription factor CHOP (CCAAT-binding homologous protein; GADD 153) is a critical cellular response for the transcriptional control of endoplasmic reticulum (ER) stress-induced apoptosis. Upon nuclear translocation, CHOP upregulates the transcription of proapoptotic factors and downregulates antiapoptotic genes. Transcriptional activation by CHOP involves heterodimerization with other members of the basic leucine zipper transcription factor (bZIP) family. We show that the bZIP protein C/EBP beta isoform LIP is required for nuclear translocation of CHOP during ER stress. In early ER stress, LIP undergoes proteasomal degradation in the cytoplasmic compartment. During later ER stress, LIP binds CHOP in both cytoplasmic and nuclear compartments and contributes to its nuclear import. By using CHOP-deficient cells and transfections of LIP-expressing vectors in C/EBP beta(-/-) mouse embryonic fibroblasts (MEFs), we show that the LIP-CHOP interaction has a stabilizing role for LIP. At the same time, CHOP uses LIP as a vehicle for nuclear import. LIP-expressing C/EBP beta(-/-) MEFs showed enhanced ER stress-induced apoptosis compared to C/EBP beta-null cells, a finding in agreement with the decreased levels of Bcl-2, a known transcriptional control target of CHOP. In view of the positive effect of CHOP-LIP interaction in mediating their proapoptotic functions, we propose this functional cooperativity as molecular symbiosis between proteins.

Amino acid starvation induces the SNAT2 neutral amino acid transporter by a mechanism that involves eukaryotic initiation factor 2alpha phosphorylation and cap-independent translation.

Nutritional stress caused by amino acid starvation involves a coordinated cellular response that includes the global decrease of protein synthesis and the increased production of cell defense proteins. Part of this response is the induction of transport system A for neutral amino acids that leads to the recovery of cell volume and amino acid levels once extracellular amino acid availability is restored. Hypertonic stress also increases system A activity as a mechanism to promote a rapid recovery of cell volume. Both a starvation-dependent and a hypertonic increase of system A transport activity are due to the induction of SNAT2, the ubiquitous member of SLC38 family. The molecular mechanisms underlying SNAT2 induction were investigated in tissue culture cells. We show that the increase in system A transport activity and SNAT2 mRNA levels upon amino acid starvation were blunted in cells with a mutant eIF2alpha that cannot be phosphorylated. In contrast, the induction of system A activity and SNAT2 mRNA levels by hypertonic stress were independent of eIF2alpha phosphorylation. The translational control of the SNAT2 mRNA during amino acid starvation was also investigated. It is shown that the 5'-untranslated region contains an internal ribosome entry site that is constitutively active in amino acid-fed and -deficient cells and in a cell-free system. We also show that amino acid starvation caused a 2.5-fold increase in mRNA and protein expression from a reporter construct containing both the SNAT2 intronic amino acid response element and the SNAT2-untranslated region. We conclude that the adaptive response of system A activity to amino acid starvation requires eukaryotic initiation factor 2alpha phosphorylation, increased gene transcription, and internal ribosome entry site-mediated translation. In contrast, the response to hypertonic stress does not involve eukaryotic initiation factor 2alpha phosphorylation, suggesting that SNAT2 expression can be modulated by specific signaling pathways in response to different stresses.