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Background: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with skin barrier defects and a misdirected type 2 immune response against harmless antigens. The skin microbiome in AD is characterized by a reduction in microbial diversity with a dominance of staphylococci, including Staphylococcus epidermidis (S. epidermidis). Objective: To assess whether S. epidermidis antigens play a role in AD, we screened for candidate allergens and studied the T cell and humoral immune response against the extracellular serine protease (Esp). Methods: To identify candidate allergens, we analyzed the binding of human serum IgG4, as a surrogate of IgE, to S. epidermidis extracellular proteins using 2-dimensional immunoblotting and mass spectrometry. We then measured serum IgE and IgG1 binding to recombinant Esp by ELISA in healthy and AD individuals. We also stimulated T cells from AD patients and control subjects with Esp and measured the secreted cytokines. Finally, we analyzed the proteolytic activity of Esp against IL-33 and determined the cleavage sites by mass spectrometry. Results: We identified Esp as the dominant candidate allergen of S. epidermidis. Esp-specific IgE was present in human serum; AD patients had higher concentrations than controls. T cells reacting to Esp were detectable in both AD patients and healthy controls. The T cell response in healthy adults was characterized by IL-17, IL-22, IFN-γ, and IL-10, whereas the AD patients’ T cells lacked IL-17 production and released only low amounts of IL-22, IFN-γ, and IL-10. In contrast, Th2 cytokine release was higher in T cells from AD patients than from healthy controls. Mature Esp cleaved and activated the alarmin IL-33. Conclusion: The extracellular serine protease Esp of S. epidermidis can activate IL-33. As an antigen, Esp elicits a type 2-biased antibody and T cell response in AD patients. This suggests that S. epidermidis can aggravate AD through the allergenic properties of Esp. Graphical abstract [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen. Author summary: The uptake of diverse oligopeptides enables pneumococcal growth despite auxotrophies and functions as a critical sensor for assessing the composition of the local environment. The identification of additional OBPs in non-encapsulated S. pneumoniae strains suggests their involvement in sensing a broader spectrum of bacterial competitors coexisting with the highly commensal pneumococcus. This study presents a comprehensive analysis of the initial phase of peptide transport mediated by OBPs within the pneumococcal Ami permease system. We disclose a common mechanism for oligopeptide recognition that is modulated in each OBP to accommodate a diverse array of oligopeptides. Understanding how pneumococcus perceives external stimuli and responds to them is imperative for unraveling the transition from a commensal to a pathogenic state. [ABSTRACT FROM AUTHOR]

In its natural habitats, Bacillus subtilis is exposed to changing osmolarity, necessitating adaptive stress responses. Transcriptomic and proteomic approaches can provide a picture of the dynamic changes occurring in salt-stressed B. subtilis cultures because these studies provide an unbiased view of cells coping with high salinity. We applied whole-genome microarray technology and metabolic labeling, combined with state-of-theart proteomic techniques, to provide a global and time-resolved picture of the physiological response of B. subtilis cells exposed to a severe and sudden osmotic upshift. This combined experimental approach provided quantitative data for 3,961 mRNA transcription profiles, 590 expression profiles of proteins detected in the cytosol, and 383 expression profiles of proteins detected in the membrane fraction. Our study uncovered a well-coordinated induction of gene expression subsequent to an osmotic upshift that involves large parts of the SigB, SigW, SigM, and SigX regulons. Additionally osmotic upregulation of a large number of genes that do not belong to these regulons was observed. In total, osmotic upregulation of about 500 B. subtilis genes was detected. Our data provide an unprecedented rich basis for further in-depth investigation of the physiological and genetic responses of B. subtilis to hyperosmotic stress. doi: 10.1128/JB.01106-09

The synthesis of structural components and morphogenetic factors required for the assembly of the Bacillus subtilis spore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma [[sigma.sup.E] factor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on [[sigma].sup.K], whose activation follows engulfment completion, and GerE. The cotE gene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme, cotE is expressed first from a [[sigma].sup.E]-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under [[sigma.sup.k] control. Cote localizes prior to engulfment completion close to the surface of the developing spore, hut formation of the outer coat is a late, [[sigma].sup.K]-controlled event. We have transplanted cotE to progressively later classes of mother cell gene expression. This created an early class of mutants in which cotE is expressed prior to engulfment completion and a late class in which expression of cotE follows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of Cote is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that Cote has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly.

The soil bacterium Bacillus subtilis frequently encounters a reduction in temperature in its natural habitats. Here, a combined transcriptomic and proteomic approach has been used to analyse the adaptational responses of B. subtilis to low temperature. Propagation of B. subtilis in minimal medium at 15[degrees]C triggered the induction of 279 genes and the repression of 301 genes in comparison to cells grown at 37[degrees]C. The analysis thus revealed profound adjustments in the overall gene expression profile in chill-adapted cells. Important transcriptional changes in low-temperature-grown cells comprise the induction of the SigB-controlled general stress regulon, the induction of parts of the early sporulation regulons (SigF, SigE and SigG) and the induction of a regulatory circuit (RapA/PhrA and Opp) that is involved in the fine-tuning of the phosphorylation status of the Spo0A response regulator. The analysis of chill-stress-repressed genes revealed reductions in major catabolic (glycolysis, oxidative phosphorylation, ATP synthesis) and anabolic routes (biosynthesis of purines, pyrimidines, haem and fatty acids) that likely reflect the slower growth rates at low temperature. Low-temperature repression of part of the SigW regulon and of many genes with predicted functions in chemotaxis and motility was also noted. The proteome analysis of chill-adapted cells indicates a major contribution of post-transcriptional regulation phenomena in adaptation to low temperature. Comparative analysis of the previously reported transcriptional responses of cold-shocked B. subtilis cells with this data revealed that cold shock and growth in the cold constitute physiologically distinct phases of the adaptation of B. subtilis to low temperature.

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains [epsilon]-([gamma]-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under [[sigma].sup.K] control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.

Temporal and compartment-specific control of gene expression during sporulation in Bacillus subtilis is governed by a cascade of four RNA polymerase subunits. [[sigma].sup.F] in the prespore and [[sigma].sup.E] in the mother cell control early stages of development, and are replaced at later stages by [[sigma].sup.G] and [[sigma].sup.K], respectively. Ultimately, a comprehensive description of the molecular mechanisms underlying spore morphogenesis requires the knowledge of all the intervening genes and their assignment to specific regulons. Here, in an extension of earlier work, DNA macroarrays have been used, and members of the four compartment-specific sporulation regulons have been identified. Genes were identified and grouped based on: i) their temporal expression profile and ii) the use of mutants for each of the four sigma factors and a bofA allele, which allows [[sigma].sup.K] activation in the absence of [[sigma].sup.G]. As a further test, artificial production of active alleles of the sigma factors in non-sporulating cells was employed. A total of 439 genes were found, including previously characterized genes whose transcription is induced during sporulation: 55 in the [[sigma].sup.F] regulon, 154 [[sigma].sup.E]-governed genes, 113 [[sigma].sup.G]-dependent genes, and 132 genes under [[sigma].sup.K] control. The results strengthen the view that the activities of [[sigma].sup.F], [[sigma].sup.E], [[sigma].sup.G] and [[sigma].sup.K] are largely compartmentalized, both temporally as well as spatially, and that the major vegetative sigma factor ([[sigma].sup.R]) is active throughout sporulation. The results provide a dynamic picture of the changes in the overall pattern of gene expression in the two compartments of the sporulating cell, and offer insight into the roles of the prespore and the mother cell at different times of spore morphogenesis.

General stress proteins protect Bacillus subtilis cells against a variety of environmental insults. This adaptive response is particularly important for nongrowing cells, to which it confers a multiple, nonspecific, and preemptive stress resistance. Induction of the general stress response relies on the alternative transcription factor, SigB, whose activity is controlled by a partner switching mechanism that also involves the anti-sigma factor, RsbW, and the antagonist protein, RsbV. Recently, the SigB regulon has been shown to be continuously induced and functionally important in cells actively growing at low temperature. With the exception of this chill induction, all SigB-activating stimuli identified so far trigger a transient expression of the SigB regulon that depends on RsbV. Through a proteome analysis and Northern blot and gene fusion experiments, we now show that the SigB regulon is continuously induced in cells growing actively at 51 [degrees] C, close to the upper growth limit of B. subtilis. This heat induction of SigB-dependent genes requires the environmental stress-responsive phosphatase RsbU, but not the metabolic stress-responsive phosphatase RsbP. RsbU dependence of SigB activation by heat is overcome in mutants that lack RsbV. In addition, loss of RsbV alone or in combination with RsbU triggers a hyperactivation of the general stress regulon exclusively at high temperatures detrimental for cell growth. These new facets of heat induction of the SigB regulon indicate that the current view of the complex genetic and biochemical regulation of SigB activity is still incomplete and that SigB perceives signals independent of the RsbV-mediated signal transduction pathways under heat stress conditions.

Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the B. subtilis oxdD gene was previously shown to have oxalate decarboxylase activity when it was produced in Escherichia coli and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of oxdD is induced during sporulation as a monocistronic unit under the control of [[sigma].sup.K] and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a cotE mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of oxdD resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.

The gram-positive soil bacterium Bacillus subtilis often faces increases in the salinity in its natural habitats. A transcriptional profiling approach was utilized to investigate both the initial reaction to a sudden increase in salinity elicited by the addition of 0.4 M NaCl and the cellular adaptation reactions to prolonged growth at high salinity (1.2 M NaCl). Following salt shock, a sigB mutant displayed immediate and transient induction and repression of 75 and 51 genes, respectively. Continuous propagation of this strain in the presence of 1.2 M NaCl triggered the induction of 123 genes and led to the repression of 101 genes. In summary, our studies revealed (i) an immediate and transient induction of the SigW regulon following salt shock, (ii) a role of the DegS/DegU two-component system in sensing high salinity, (iii) a high-salinity-mediated iron limitation, and (iv) a repression of chemotaxis and motility genes by high salinity, causing severe impairment of the swarming capability of B. subtilis cells. Initial adaptation to salt shock and continuous growth at high salinity share only a limited set of induced and repressed genes. This finding strongly suggests that these two phases of adaptation require distinctively different physiological adaptation reactions by the B. subtilis cell. The large portion of genes with unassigned functions among the high-salinity-induced or -repressed genes demonstrates that major aspects of the cellular adaptation of B. subtilis to high salinity are unexplored so far.

Summary: The Gram‐positive bacterium Bacillus subtilis is frequently exposed to hyperosmotic conditions. In addition to the induction of genes involved in the accumulation of compatible solutes, high salinity exerts widespread effects on B. subtilis physiology, including changes in cell wall metabolism, induction of an iron limitation response, reduced motility and suppression of sporulation. We performed a combined whole‐transcriptome and proteome analysis of B. subtilis 168 cells continuously cultivated at low or high (1.2 M NaCl) salinity. Our study revealed significant changes in the expression of more than one‐fourth of the protein‐coding genes and of numerous non‐coding RNAs. New aspects in understanding the impact of high salinity on B. subtilis include a sustained low‐level induction of the SigB‐dependent general stress response and strong repression of biofilm formation under high‐salinity conditions. The accumulation of compatible solutes such as glycine betaine aids the cells to cope with water stress by maintaining physiologically adequate levels of turgor and also affects multiple cellular processes through interactions with cellular components. Therefore, we additionally analysed the global effects of glycine betaine on the transcriptome and proteome of B. subtilis and revealed that it influences gene expression not only under high‐salinity, but also under standard growth conditions. [ABSTRACT FROM AUTHOR]

Background: Despite observational evidence from epidemiological and clinical studies associating sex hormones with various cardiometabolic risk factors or diseases, pathophysiological explanations are sparse to date. To reveal putative functional insights, we analyzed associations between sex hormone levels and whole blood gene expression profiles. Methods: We used data of 991 individuals from the population-based Study of Health in Pomerania (SHIP-TREND) with whole blood gene expression levels determined by array-based transcriptional profiling and serum concentrations of total testosterone (TT), sex hormone-binding globulin (SHBG), free testosterone (free T), dehydroepiandrosterone sulfate (DHEAS), androstenedione (AD), estradiol (E2), and estrone (E1) measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay. Associations between sex hormone concentrations and gene expression profiles were analyzed using sex-specific regression models adjusted for age, body mass index, and technical covariables. Results: In men, positive correlations were detected between AD and DDIT4 mRNA levels, as well as between SHBG and the mRNA levels of RPIA, RIOK3, GYPB, BPGM, and RAB2B. No additional significant associations were observed. Conclusions: Besides the associations between AD and DDIT4 expression and SHBG and the transcript levels of RPIA, RIOK3, GYPB, BPGM, and RAB2B, the present study did not indicate any association between sex hormone concentrations and whole blood gene expression profiles in men and women from the general population. [ABSTRACT FROM AUTHOR]

Heart failure induced by myocardial infarction (MI) causes diaphragm muscle weakness, with elevated oxidants implicated. We aimed to determine whether diaphragm muscle weakness is 1) early-onset post-MI (i.e., within the early left ventricular remodeling phase of 72 h); and 2) associated with elevated protein oxidation. Ligation of the left coronary artery to induce MI (n = 10) or sham operation (n = 10) was performed on C57BL6 mice. In vitro contractile function of diaphragm muscle fiber bundles was assessed 72 h later. Diaphragm mRNA and protein expression, enzyme activity, and individual car-bonylated proteins (by two-dimensional differential in-gel electropho-resis and mass spectrometry) were subsequently assessed. Infarct size averaged 57 ± 1%. Maximal diaphragm function was reduced (P < 0.01) by 20% post-MI, with the force-frequency relationship depressed (P < 0.01) between 80 and 300 Hz. The mRNA expression of inflammation, atrophy, and regulatory Ca2+ proteins remained unchanged post-MI, as did the protein expression of key contractile proteins. However, enzyme activity of the oxidative sources NADPH oxidase and xanthine oxidase was increased (P < 0.01) by 45 and 33%, respectively. Compared with sham, a 57 and 45% increase (P < 0.05) was observed in the carbonylation of sarcomeric actin and creatine kinase post-MI, respectively. In conclusion, diaphragm muscle weakness was rapidly induced in mice during the early left ventricular remodeling phase of 72 h post-MI, which was associated with increased oxidation of contractile and energetic proteins. Collectively, these findings suggest diaphragm muscle weakness may be early onset in heart failure, which is likely mediated in part by posttranslational oxidative modifications at the myofibrillar level. [ABSTRACT FROM AUTHOR]

The Gram-positive bacterium B acillus subtilis encounters nutrient limitations and osmotic stress in its natural soil ecosystem. To ensure survival and sustain growth, highly integrated adaptive responses are required. Here, we investigated the system-wide response of B . subtilis to different, simultaneously imposed stresses. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses of the transcriptome, proteome, metabolome and fluxome. Surprisingly, the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. Specifically, increased production of several enzymes of central carbon metabolism compensates for their reduced activity in the presence of high salt. A major response of the cell during osmotic stress is the production of the compatible solute proline. This is achieved through the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways. [ABSTRACT FROM AUTHOR]

Background: A.BY/SnJ mice are used to study pathological alterations in the heart due to enteroviral infections. Since age is a well-known factor influencing the susceptibility of mice to infection, response to stress and manifestation of cardiovascular diseases, the myocardial proteome of A.BY/SnJ mice aged 1 and 4 months was comparatively studied using two dimensional-differential in-gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: Complementary analyses by 2D-DIGE and gel-free LC-MS/MS revealed 96 distinct proteins displaying age associated alterations in their levels. Proteins related to protein transport, and transport chain, lipid metabolism and fatty acid transport showed significant changes in 4 months old mouse hearts compared to juvenile hearts. Proteins involved in lipid metabolism and transport were identified at significantly higher levels in older mice and dysregulation of proteins of the respiratory transport chain were observed. Conclusion: The current proteomics study discloses age dependent changes occurring in the hearts already in young mice of the strain A.BY/SnJ. Besides alterations in protein transport, we provide evidence that a decrease of ATP synthase in murine hearts starts already in the first months of life, leading to well-known low expression levels manifested in old mice thereby raising the possibility of reduced energy supply. In the first few months of murine life this seems to be compensated by an increased lipid metabolism. The functional alterations described should be considered during experimental setups in disease related studies. [ABSTRACT FROM AUTHOR]