Adaptive Response of Group B Streptococcus to High Glucose Conditions: New Insights on the CovRS Regulation Network

Although the contribution of carbohydrate catabolism to bacterial colonization and infection is well recognized, the transcriptional changes during these processes are still unknown. In this study, we have performed comparative global gene expression analysis of GBS in sugar-free versus high glucose milieu. The analysis revealed a differential expression of genes involved in metabolism, transport and host-pathogen interaction. Many of them appeared to be among the genes previously reported to be controlled by the CovRS two-component system. Indeed, the transcription profile of a Delta covRS strain grown in high-glucose conditions was profoundly affected. In particular, of the total genes described to be regulated by glucose, similar to 27% were under CovRS control with a functional role in protein synthesis, transport, energy metabolism and regulation. Among the CovRS dependent genes, we found bibA, a recently characterized adhesin involved in bacterial serum resistance and here reported to be down-regulated by glucose. ChIP analysis revealed that in the presence of glucose, CovR binds bibA promoter in vivo, suggesting that CovR may act as a negative regulator or a repressor. We also demonstrated that, as for other target promoters, chemical phosphorylation of CovR in aspartic acid increases its affinity for the bibA promoter region. The data reported in this study contribute to the understanding of the molecular mechanisms modulating the adaptation of GBS to glucose

A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA expression, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract of the nadA promoter gene and contributes to the differential expression levels of phase variant promoters with different numbers of repeats, likely due to different spacing between operators. It is shown that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces nadA expression by inhibiting the DNA binding activity of the NadR repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants, which are likely due to differential RNA polymerase contacts leading to altered promoter activity. These results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, and both regulations are mediated by the NadR repressor that and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals

The RNA chaperone Hfq is involved in the stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression

The well conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. It is thought to function as an RNA chaperone and facilitate base-pairing between small regulatory RNA (sRNA) and the mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis we generated a Hfq mutant of the MC58 strain and the knockout mutant shows pleiotropic phenotypes: it has a general growth phenotype in vitro in culture media, it is sensitive to a wide range of stresses including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence and proposes a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which is as yet largely unexplored

Pseudomonas aeruginosa outer membrane vesicles triggered by human mucosal fluid and lysozyme can prime host tissue surfaces for bacterial adhesion

Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release Outer Membrane Vesicles (OMVs) in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to PBS controls (~100 fold). TEM and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (~4-fold, P < 0.01). Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections

Real-time RT-PCR evaluation of <i>bibA</i> expression in 2603V/R and Δc<i>ovRS</i> strains grown in medium containing 55 mM glucose or in sugar-free medium.

<p>Transcript levels were normalized to the expression level of <i>gyrA</i>. Syber green runs were performed with cDNAs from the same reverse transcription reaction from 1 µg of total RNA. The ΔΔCT method was applied as a comparative method of quantification, using strains grown in sugar free medium as reference. The data are representative of 2 independent experiments, each in triplicate. Error bars, SD.</p

Transcriptome changes elicited by glucose in GBS.

<p>Comparison of gene expression changes (log2) between mid-log cultures of 2603 V/R wild-type strain and <i>CovRS</i> mutant following challenge with or without glucose. Data for the wild type and mutant strains are shown on the <i>x</i> axis and <i>y</i> axis, respectively.</p

CovR binds to <i>bibA</i> promoter <i>in vivo</i>. (A)

<p>Quantification by qRT-PCR of <i>bibA</i> promoter immunoprecipitated with CovR antiserum in 2603 V/R wild type strain grown in medium devoid of glucose or in the presence of 55mM glucose. <i>cfb</i> promoter and <i>cylX</i> promoter were used as a positive control while <i>sag0017</i> promoter was used as a negative control. The level of PCR products of eluate from the isogenic Δc<i>ovRS</i> deletion mutant grown with or without glucose was negligible. The data are representative of 3 independent experiments, each in triplicate. Error bars, SD. (<b>B)</b> Competitive EMSA experiment. Labelled <i>PbibA</i> fragment (3.3 nM) was incubated without <i>(lane1)</i> or with CovR (2 µM) <i>(lane2</i>–<i>6)</i>, in the presence of different amounts of unlabelled <i>PbibA (lane 3</i>–<i>4)</i>, as a specific competitor, and <i>Psag0017 (lane5</i>–<i>6)</i>, as a non-specific competitor. The labelled DNA was detected by chemioluminescence. <b>(C)</b> CovR phosphorylation increases its affinity for <i>bibA</i> promoter. Electrophoretic mobility shift assay using recombinant CovR (left) and chemically phosphorylated recombinant CovR (right). Labelled <i>PbibA</i> DNA fragment (3.3 nM) was incubated without or with the indicated amounts of CovR. The labelled DNA was detected by chemioluminescence.</p

Differential regulation of gene expression in GBS strain 2603 V/R versus the isogenic Δ<i>CovRS</i> mutant strain after incubation in medium with 55 mM glucose versus a sugars-free complex medium.

<p>White bars indicate the number of glucose-regulated genes in the wild-type strain; black bars indicate the number of genes that are glucose- dependent and CovRS-dependent; grey bars indicate the number of genes that are glucose-dependent and CovRS-independent.</p

List of genes highly regulated in GBS strain 2603V/R after incubation in high glucose medium.

<p>List of genes highly regulated in GBS strain 2603V/R after incubation in high glucose medium.</p

Graphical representation summarizing adaptive regulation of GBS in high glucose conditions.

<p>Genes of interest are color-grouped according to main functional categories. Arrows indicate up- or down-regulation relative to time of 30′ in high glucose <i>vs.</i> no glucose.</p