Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors

Enterococcus faecalis V583 is a vancomycin-resistant clinical isolate which belongs to the hospital-adapted clade, CC2. This strain harbours several factors that have been associated with virulence, including the fsr quorum-sensing regulatory system that is known to control the expression of GelE and SprE proteases. To discriminate between genes directly regulated by Fsr, and those indirectly regulated as the result of protease expression or activity, we compared gene expression in isogenic mutants of V583 variously defective in either Fsr quorum sensing or protease expression. Quorum sensing was artificially induced by addition of the quorum signal, GBAP, exogenously in a controlled manner. The Fsr regulon was found to be restricted to five genes, gelE, sprE, ef1097, ef1351 and ef1352. Twelve additional genes were found to be dependent on the presence of GBAP-induced proteases. Induction of GelE and SprE by GBAP via Fsr resulted in accumulation of mRNA encoding lrgAB, and this induction was found to be lytRS dependent. Drosophila infection was used to discern varying levels of toxicity stemming from mutations in the fsr quorum regulatory system and the genes that it regulates, highlighting the contribution of LrgAB and bacteriocin EF1097 to infection toxicity. A contribution of SprE to infection toxicity was also detected. This work brought to light new players in E. faecalis success as a pathogen and paves the way for future studies on host tolerance mechanisms to infections caused by this important nosocomial pathogen

Cytomegaloviral determinants of CD8+ T cell programming and RhCMV/SIV vaccine efficacy

Simian immunodeficiency virus (SIV) insert-expressing, 68–1 Rhesus Cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex (MHC)-E- and -II-restricted, SIV-specific CD8(+) T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) has not been characterized. We show that these unconventional responses resulted from a chance genetic rearrangement in 68–1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions (Rh157.5/Rh157.4 and Rh158–161), revealing three patterns of unconventional response inhibition. Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)-derived sequences (UL128/UL130; UL146/UL147) leads to either of two distinct CD8(+) T cell response types – MHC-Ia-restricted-only, or a mix of MHC-II- and MHC-Ia-restricted CD8(+) T cells. Response magnitude and functional differentiation are similar to RhCMV 68–1, but neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E-restricted CD8(+) T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all genes that inhibit these responses from the HCMV/HIV vector

Strains, plasmids and primers used in this study.

<p>Strains, plasmids and primers used in this study.</p

<i>Drosophila</i> Host Model Reveals New <i>Enterococcus faecalis</i> Quorum-Sensing Associated Virulence Factors

<div><p><i>Enterococcus faecalis</i> V583 is a vancomycin-resistant clinical isolate which belongs to the hospital-adapted clade, CC2. This strain harbours several factors that have been associated with virulence, including the <i>fsr</i> quorum-sensing regulatory system that is known to control the expression of GelE and SprE proteases. To discriminate between genes directly regulated by Fsr, and those indirectly regulated as the result of protease expression or activity, we compared gene expression in isogenic mutants of V583 variously defective in either Fsr quorum sensing or protease expression. Quorum sensing was artificially induced by addition of the quorum signal, GBAP, exogenously in a controlled manner. The Fsr regulon was found to be restricted to five genes, <i>gelE</i>, <i>sprE</i>, <i>ef1097</i>, <i>ef1351</i> and <i>ef1352</i>. Twelve additional genes were found to be dependent on the presence of GBAP-induced proteases. Induction of GelE and SprE by GBAP via Fsr resulted in accumulation of mRNA encoding <i>lrgAB</i>, and this induction was found to be <i>lytRS</i> dependent. <i>Drosophila</i> infection was used to discern varying levels of toxicity stemming from mutations in the <i>fsr</i> quorum regulatory system and the genes that it regulates, highlighting the contribution of LrgAB and bacteriocin EF1097 to infection toxicity. A contribution of SprE to infection toxicity was also detected. This work brought to light new players in <i>E. faecalis</i> success as a pathogen and paves the way for future studies on host tolerance mechanisms to infections caused by this important nosocomial pathogen.</p></div

GBAP-dependent regulatory network.

<p>Once the GBAP (black disks) concentration outside cells reaches a certain threshold (upper part of the cell), the Fsr system is activated, and the FsrA regulator induces expression of <i>gelE</i>, <i>sprE</i> and <i>ef1097</i> genes. Both produce proteins which will be located to the cell membrane and cell wall. Although GelE is loosely bound to the cell, it will also be released from it. The induced expression of <i>ef1352</i>, which encodes a putative MgtA protein, by GBAP is likely due to increased amounts of EF1097, predicted to be a bacteriocin. EF1352 could function as an auto-immunity factor against EF1097. The increased level of GelE and SprE proteins in the cell-wall in response to GBAP are proposed to induce changes sensed by LytS protein, which in turn, activates LytR, responsible for induction of <i>lrgAB</i> genes. When no GBAP is produced (lower part of the cell) <i>ef1097</i> is not expressed, but both GelE and SprE are still produced, although in lower amounts (dotted line). In this situation, <i>lrgAB</i> genes are still expressed, but the increment in their expression during growth in the exponential phase (assayed during microarrays performed without GBAP) is not due to the QS molecule. As we found that <i>lrgAB</i> can still be expressed in a <i>lytRS</i> mutant, we propose that this is not the only regulator able to induce expression of that operon.</p

Genes differentially expressed upon addition of GBAP to V583<i>ΔfsrB</i>, V583<i>ΔfsrBΔgelE</i>, V583<i>ΔfsrBΔsprE</i> and V583<i>ΔfsrBΔgelEΔsprE</i> strains.

<p>Fold-change values were obtained by comparing gene expression at 10 min against 0 min post-GBAP addition, by microarray analysis.</p>1<p>Fold-change values were obtained by comparing gene expression at 10 min against 0 min post-GBAP addition, by microarray analysis. (+) up-regulated (−) down-regulated;</p>2<p>These two genes were up-regulated in the experiments done without GBAP, only in the V583<i>ΔfsrB</i> strain with a fold change of +7 for E3193 and +6 for EF3194;</p>3<p><i>ef0411</i> is part of the predicted operon <i>ef0411-0412-0413</i>, which encodes a mannitol specific PTS-system;</p>4<p>LemA-like protein likely involved in cell wall metabolism. LemA proteins contain a predicted amino terminal transmembrane helix and a short extracellular amino terminus. The exact molecular function of this protein is uncertain;</p>5<p>Has two predicted transmembrane helixes and a Blast search does not reveal similarity to proteins of known function. Upstream is a putative operon encoding the potassium-transporting ATPase KdpABC (EF0567–EF0569) and the two-component system KdpED (EF0570–EF0571) (TCS12) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064740#pone.0064740-Hancock2" target="_blank">[62]</a>;</p>6<p>It has a predicted transmembrane domain at its N-terminus (residues 4 to 20) and the rest of the protein is located outside the cell. It has a predicted thioredoxin fold domain similar to bacteriocin accessory proteins ((http: //<a href="http://www.genome.jp/dbget-bin/www_bget?efa" target="_blank">www.genome.jp/dbget-bin/www_bget?efa</a>: EF0776);</p>7<p>Predicted to facilitate the conversion of aspartate and alpha-ketoglutarate to oxaloacetate and glutamate;</p>8<p>Part of the predicted operon <i>ef1218–ef1224</i>, which codes for a spermidine/putrescine ABC transporter;</p>9<p>EF1815 has 25% amino acid sequence similarity to CidR from <i>S. aureus</i> (http: //blast.ncbi.nlm.nih.gov/); EF1816 is a hypothetical protein with a β-lactamase domain, has no transmembrane domain, and is orthologous to PhnP, which is involved in phosphonate metabolism. EF1815 and EF1816 are located upstream of SprE (EF1817), but only EF1816 is located in the positive DNA strand.</p

LytRS is required for GBAP induction of <i>lrgAB</i> genes.

<p>The semi-quantitative RT-PCR shows expression of <i>lrgAB</i> genes in the VI13 (Δ<i>fsrB</i> mutant) and KS19 (Δ<i>fsrB</i>Δ<i>lytRS</i> mutant), in the presence of GBAP. Expression of <i>gelE</i> and <i>gdh</i> were used as positive and negative controls, respectively, of Fsr induction by GBAP and of RNA concentration, respectively. The RNA used for this analysis was previously treated with RNase-free DNase I to remove contaminating DNA.</p

<i>Drosophila</i> survival rates upon infection with <i>E. faecalis</i> strains.

<p>75 Oregon R (5- to 7-day-old) male adult flies, raised at 25°C, were divided in tubes of 25 flies each, and infected, by septic injury onto the thorax with a thin needle, with V583 (A, B, D) and VE14089 derived strains (C). Data is representative of three independent experiments (225 flies per strain). Curves assigned with an * are significantly different (p<0.0001) from the respective wild-type -infected curve, as determined by log-rank analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064740#pone.0064740.s004" target="_blank">Table S2</a>).</p