Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e σB regulon

<p>Abstract</p> <p>Background</p> <p>The opportunistic food-borne gram-positive pathogen <it>Listeria monocytogenes </it>can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, σ^B, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine σ^Bexpression during growth of <it>L. monocytogenes </it>EGD-e. Whole genome-based transcriptional profiling was used to identify σ^B-dependent genes at different growth phases.</p> <p>Results</p> <p>We detected 105 σ^B-positively regulated genes and 111 genes which appeared to be under negative control of σ^Band validated 36 σ^B-positively regulated genes <it>in vivo </it>using a reporter gene fusion system.</p> <p>Conclusion</p> <p>Genes comprising the σ^Bregulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the σ^B-positively regulated genes of <it>L. monocytogenes </it>are absent in <it>L. innocua</it>. A total of 75 σ^B-positively regulated listerial genes had homologs in <it>B. subtilis</it>, but only 33 have been previously described as being σ^B-regulated in <it>B. subtilis </it>even though both species share a highly conserved σ^B-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.</p

Adaptive immune response to lipoproteins of Staphylococcus aureus in healthy subjects

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life-threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll-like receptors 2 and 6. This study addressed the specific B-cell and T-cell responses directed to lipoproteins in human S. aureus carriers and non-carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells.</p

Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A

Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability

Take Your Child to Conferences? On the Need for Family-Friendly Policies During DGPS Section Clinical Psychology Conferences

Reconciling science and family is a major challenge for parents, particularly for women. Attending conferences is important to one's career but an organizational challenge for parents. In this paper, we surveyed the opinion of the German Psychological Society's (DGP's) Division of Clinical Psychology and Psychotherapy on family-friendly conference arrangements. 147 members (response rate: 18.36%) answered questions about demographics. attitudes toward the division's conference. care options, and family-friendly arrangements. Of the participants, 66% were parents, and 45% had canceled their attendance at a conference at least once because of family obligations. Additional costs were considered high, and more family-friendly policies were desired by many participants. Family-friendly conferences can send a strong signal of inclusion and solidarity, and can ensure the maintenance and sustainability of scientific competence

Global analysis of the impact of linezolid onto virulence factor production in S. aureus USA300

The translation inhibitor linezolid is an antibiotic of last resort against Gram-positive pathogens including methicillin resistant strains of the nosocomial pathogen Staphylococcus aureus. Linezolid is reported to inhibit production of extracellular virulence factors, but the molecular cause is unknown. To elucidate the physiological response of S. aureus to linezolid in general and the inhibition of virulence factor synthesis in particular a holistic study was performed. Linezolid was added to exponentially growing S. aureus cells and the linezolid stress response was analyzed with transcriptomics and quantitative proteomics methods. In addition, scanning and transmission electron microscopy experiments as well as fluorescence microscopy analyses of the cellular DNA and membrane were performed. As previously observed in studies on other translation inhibitors, S. aureus adapts its protein biosynthesis machinery to the reduced translation efficiency. For example the synthesis of ribosomal proteins was induced. Also unexpected results like a decline in the amount of extracellular and membrane proteins were obtained. In addition, cell shape and size changed after linezolid stress and cell division was diminished. Finally, the chromosome was condensed after linezolid stress and lost contact to the membrane. These morphological changes cannot be explained by established theories. A new hypothesis is discussed, which suggests that the reduced amount of membrane and extracellular proteins and observed defects in cell division are due to the disintegration of transertion complexes by linezolid. (C) 2016 Elsevier GmbH. All rights reserved

<i>Aureolib</i> — A Proteome Signature Library: Towards an Understanding of <i>Staphylococcus aureus</i> Pathophysiology

<div><p>Gel-based proteomics is a powerful approach to study the physiology of <i>Staphylococcus aureus</i> under various growth restricting conditions. We analyzed 679 protein spots from a reference 2-dimensional gel of cytosolic proteins of <i>S. aureus</i> COL by mass spectrometry resulting in 521 different proteins. 4,692 time dependent protein synthesis profiles were generated by exposing <i>S. aureus</i> to nine infection-related stress and starvation stimuli (H₂O₂, diamide, paraquat, NO, fermentation, nitrate respiration, heat shock, puromycin, mupirocin). These expression profiles are stored in an online resource called <i>Aureolib</i> (<a href="http://www.aureolib.de" target="_blank">http://www.aureolib.de</a>). Moreover, information on target genes of 75 regulators and regulatory elements were included in the database. Cross-comparisons of this extensive data collection of protein synthesis profiles using the tools implemented in <i>Aureolib</i> lead to the identification of stress and starvation specific marker proteins. Altogether, 226 protein synthesis profiles showed induction ratios of 2.5-fold or higher under at least one of the tested conditions with 157 protein synthesis profiles specifically induced in response to a single stimulus. The respective proteins might serve as marker proteins for the corresponding stimulus. By contrast, proteins whose synthesis was increased or repressed in response to more than four stimuli are rather exceptional. The only protein that was induced by six stimuli is the universal stress protein SACOL1759. Most strikingly, cluster analyses of synthesis profiles of proteins differentially synthesized under at least one condition revealed only in rare cases a grouping that correlated with known regulon structures. The most prominent examples are the GapR, Rex, and CtsR regulon. In contrast, protein synthesis profiles of proteins belonging to the CodY and σ^B regulon are widely distributed. In summary, <i>Aureolib</i> is by far the most comprehensive protein expression database for <i>S. aureus</i> and provides an essential tool to decipher more complex adaptation processes in <i>S. aureus</i> during host pathogen interaction.</p></div

Stress experiments included in the proteome signature library.

1<p>CDM = chemically defined medium.</p

Regulons in <i>S. aureus</i>.

<p>Regulators or regulatory elements and the number of assigned proteins which are significantly and at least 2.5-fold induced (up) or repressed (down) on synthesis level.</p>1<p>number of corresponding proteins identified on the master gel belonging to the respective regulon.</p>a<p>source: <a href="http://regprecise.lbl.gov" target="_blank">http://regprecise.lbl.gov</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070669#pone.0070669-Novichkov2" target="_blank">[32]</a>.</p>b<p>source: Pagels <i>et al.</i> (2010) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070669#pone.0070669-Pagels1" target="_blank">[36]</a>.</p>c<p>source: Bischoff et al. 2004 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070669#pone.0070669-Bischoff1" target="_blank">[48]</a>, Pané-Farré et al. 2006 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070669#pone.0070669-PanFarr1" target="_blank">[49]</a>, Ziebandt et al. 2001 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070669#pone.0070669-Ziebandt2" target="_blank">[71]</a>, Ziebandt et al. 2004 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070669#pone.0070669-Ziebandt3" target="_blank">[72]</a>.</p

Response of <i>S. aureus</i> to H₂O₂.

<p>(<b>A</b>) <i>S. aureus</i> COL was cultivated in synthetic medium at 37°C and treated with 10 mM H₂O₂ at OD₅₀₀ 0.5 (0 min). (<b>B</b>) Protein synthesis profiles were normalized using total normalization and standardized (z-score). Significantly changed synthesis profiles were determined by ANOVA (α = 0.1, distribution based on all permutations, absolute threshold of 2.5). Based on their expression kinetics in response to H₂O₂, proteins can be allocated to three groups: proteins which are induced during the acute phase immediately after imposition of stress (red lines), proteins induced during resumption of growth (blue lines) and proteins highly expressed in the tolerance phase (green lines).</p