SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures

Repression of SPI2 transcription by nitric oxide-producing, IFNγ-activated macrophages promotes maturation of Salmonella phagosomes

By remodeling the phagosomal membrane, the type III secretion system encoded within the Salmonella pathogenicity island-2 (SPI2) helps Salmonella thrive within professional phagocytes. We report here that nitric oxide (NO) generated by IFNγ-activated macrophages abrogates the intracellular survival advantage associated with a functional SPI2 type III secretion system. NO congeners inhibit overall expression of SPI2 effectors encoded both inside and outside the SPI2 gene cluster, reflecting a reduced transcript level of the sensor kinase SsrA that governs overall SPI2 transcription. Down-regulation of SPI2 expression in IFNγ-treated macrophages does not seem to be the result of global NO cytotoxicity, because transcription of the housekeeping rpoD sigma factor remains unchanged, whereas the expression of the hmpA-encoded, NO-metabolizing flavohemoprotein is stimulated. Because of the reduced SPI2 expression, Salmonella-containing vacuoles interact more efficiently with compartments of the late endosomal/lysosomal system in NO-producing, IFNγ-treated macrophages. These findings demonstrate that inhibition of intracellular SPI2 transcription by NO promotes the interaction of Salmonella phagosomes with the degradative compartments required for enhanced antimicrobial activity. Transcriptional repression of a type III secretion system that blocks phagolysosome biogenesis represents a novel mechanism by which NO mediates resistance of IFNγ-activated phagocytes to an intracellular pathogen

Analysis of the ArcA regulon in anaerobically grown Salmonella enterica sv. Typhimurium

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) is a Gram-negative pathogen that must successfully adapt to the broad fluctuations in the concentration of dissolved dioxygen encountered in the host. In <it>Escherichia coli</it>, ArcA (Aerobic Respiratory Control) helps the cells to sense and respond to the presence of dioxygen. The global role of ArcA in <it>E. coli </it>is well characterized; however, little is known about its role in anaerobically grown <it>S</it>. Typhimurium.</p> <p>Results</p> <p>We compared the transcriptional profiles of the virulent wild-type (WT) strain (ATCC 14028s) and its isogenic <it>arcA </it>mutant grown under anaerobic conditions. We found that ArcA directly or indirectly regulates 392 genes (8.5% of the genome); of these, 138 genes are poorly characterized. Regulation by ArcA in <it>S</it>. Typhimurium is similar, but distinct from that in <it>E</it>. <it>coli</it>. Thus, genes/operons involved in core metabolic pathways (e.g., succinyl-CoA, fatty acid degradation, cytochrome oxidase complexes, flagellar biosynthesis, motility, and chemotaxis) were regulated similarly in the two organisms. However, genes/operons present in both organisms, but regulated differently by ArcA in <it>S</it>. Typhimurium included those coding for ethanolamine utilization, lactate transport and metabolism, and succinate dehydrogenases. <it>Salmonella</it>-specific genes/operons regulated by ArcA included those required for propanediol utilization, flagellar genes (<it>mcpAC</it>, <it>cheV</it>), Gifsy-1 prophage genes, and three SPI-3 genes (<it>mgtBC</it>, <it>slsA</it>, STM3784). In agreement with our microarray data, the <it>arcA </it>mutant was non-motile, lacked flagella, and was as virulent in mice as the WT. Additionally, we identified a set of 120 genes whose regulation was shared with the anaerobic redox regulator, Fnr.</p> <p>Conclusion(s)</p> <p>We have identified the ArcA regulon in anaerobically grown <it>S</it>. Typhimurium. Our results demonstrated that in <it>S</it>. Typhimurium, ArcA serves as a transcriptional regulator coordinating cellular metabolism, flagella biosynthesis, and motility. Furthermore, ArcA and Fnr share in the regulation of 120 <it>S</it>. Typhimurium genes.</p

Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages

Inactivation of [Fe-S] Metalloproteins Mediates Nitric Oxide-Dependent Killing of Burkholderia mallei

BACKGROUND: Much remains to be known about the mechanisms by which O(2)-dependent host defenses mediate broad antimicrobial activity. METHODOLOGY/PRINCIPAL FINDINGS: We show herein that reactive nitrogen species (RNS) generated by inducible nitric oxide (NO) synthase (iNOS) account for the anti-Burkholderia mallei activity of IFNgamma-primed macrophages. Inducible NOS-mediated intracellular killing may represent direct bactericidal activity, because B. mallei showed an exquisite sensitivity to NO generated chemically. Exposure of B. mallei to sublethal concentrations of NO upregulated transcription of [Fe-S] cluster repair genes, while damaging the enzymatic activity of the [Fe-S] protein aconitase. To test whether [Fe-S] clusters are critical targets for RNS-dependent killing of B. mallei, a mutation was constructed in the NO-induced, [Fe-S] cluster repair regulator iscR. Not only was the iscR mutant hypersusceptible to iNOS-mediated killing, but its aconitase pool was readily oxidized by NO donors as compared to wild-type controls. Although killed by authentic H(2)O(2), which also oxidizes [Fe-S] clusters, B. mallei appear to be resilient to NADPH oxidase-mediated cytotoxicity. The poor respiratory burst elicited by this bacterium likely explains why the NADPH oxidase is nonessential to the killing of B. mallei while it is still confined within phagosomes. CONCLUSIONS/SIGNIFICANCE: Collectively, these findings have revealed a disparate role for NADPH oxidase and iNOS in the innate macrophage response against the strict aerobe B. mallei. To the best of our knowledge, this is the first instance in which disruption of [Fe-S] clusters is demonstrated as cause of the bactericidal activity of NO congeners

Les droits disciplinaires des fonctions publiques : « unification », « harmonisation » ou « distanciation ». A propos de la loi du 26 avril 2016 relative à la déontologie et aux droits et obligations des fonctionnaires

The production of tt‾ , W+bb‾ and W+cc‾ is studied in the forward region of proton–proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98±0.02 fb−1 . The W bosons are reconstructed in the decays W→ℓν , where ℓ denotes muon or electron, while the b and c quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions.The production of $t\overline{t}$, $W+b\overline{b}$ and $W+c\overline{c}$ is studied in the forward region of proton-proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98 $\pm$ 0.02 \mbox{fb}^{-1}. The $W$ bosons are reconstructed in the decays $W\rightarrow\ell\nu$, where $\ell$ denotes muon or electron, while the $b$ and $c$ quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions

Physics case for an LHCb Upgrade II - Opportunities in flavour physics, and beyond, in the HL-LHC era

The LHCb Upgrade II will fully exploit the flavour-physics opportunities of the HL-LHC, and study additional physics topics that take advantage of the forward acceptance of the LHCb spectrometer. The LHCb Upgrade I will begin operation in 2020. Consolidation will occur, and modest enhancements of the Upgrade I detector will be installed, in Long Shutdown 3 of the LHC (2025) and these are discussed here. The main Upgrade II detector will be installed in long shutdown 4 of the LHC (2030) and will build on the strengths of the current LHCb experiment and the Upgrade I. It will operate at a luminosity up to 2×1034 cm−2s−1, ten times that of the Upgrade I detector. New detector components will improve the intrinsic performance of the experiment in certain key areas. An Expression Of Interest proposing Upgrade II was submitted in February 2017. The physics case for the Upgrade II is presented here in more depth. CP-violating phases will be measured with precisions unattainable at any other envisaged facility. The experiment will probe b → sl+l−and b → dl+l− transitions in both muon and electron decays in modes not accessible at Upgrade I. Minimal flavour violation will be tested with a precision measurement of the ratio of B(B0 → μ+μ−)/B(Bs → μ+μ−). Probing charm CP violation at the 10−5 level may result in its long sought discovery. Major advances in hadron spectroscopy will be possible, which will be powerful probes of low energy QCD. Upgrade II potentially will have the highest sensitivity of all the LHC experiments on the Higgs to charm-quark couplings. Generically, the new physics mass scale probed, for fixed couplings, will almost double compared with the pre-HL-LHC era; this extended reach for flavour physics is similar to that which would be achieved by the HE-LHC proposal for the energy frontier

LHCb upgrade software and computing : technical design report

This document reports the Research and Development activities that are carried out in the software and computing domains in view of the upgrade of the LHCb experiment. The implementation of a full software trigger implies major changes in the core software framework, in the event data model, and in the reconstruction algorithms. The increase of the data volumes for both real and simulated datasets requires a corresponding scaling of the distributed computing infrastructure. An implementation plan in both domains is presented, together with a risk assessment analysis

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder