The functions of many bacterial RNA-binding proteins remain obscure because of a lack of knowledge of their cellular ligands. Although well-studied cold-shock protein A (CspA) family members are induced and function at low temperature, others are highly expressed in infection-relevant conditions. Here, we have profiled transcripts bound in vivo by the CspA family members of Salmonella enterica serovar Typhimurium to link the constitutively expressed CspC and CspE proteins with virulence pathways. Phenotypic assays in vitro demonstrated a crucial role for these proteins in membrane stress, motility, and biofilm formation. Moreover, double deletion of cspC and cspE fully attenuates Salmonella in systemic mouse infection. In other words, the RNA ligand-centric approach taken here overcomes a problematic molecular redundancy of CspC and CspE that likely explains why these proteins have evaded selection in previous virulence factor screens in animals. Our results highlight RNA-binding proteins as regulators of pathogenicity and potential targets of antimicrobial therapy. They also suggest that globally acting RNA-binding proteins are more common in bacteria than currently appreciated.RNA-binding protein | cold-shock protein | Salmonella | bacterial pathogenesis | stress response T he myriad of coding and noncoding RNAs in a cell generally do not act in isolation but rapidly associate with RNA-binding proteins (RBPs) to execute their functions. Recent methods relying on the global capture of polyadenylated transcripts in eukaryotes have dramatically expanded our knowledge of RBP activity. These approaches have revealed many previously unsuspected RBPs, suggesting a much wider scope of posttranscriptional control based on thousands of new RBP-mRNA interactions (1, 2). Nevertheless, the functions of many of these newly discovered RBPs remain unclear.In contrast to the situation in eukaryotes, there is a paucity of knowledge about RBPs in prokaryotes. This lack of knowledge is compounded further by the lack of a poly(A) tail on functional transcripts, which precludes similar global discovery studies of bacterial RBP networks. Extensive profiling of cellular targets of the small RNA (sRNA) chaperone Hfq and the translational repressor CsrA (3-7) have shown that large posttranscriptional networks also exist in bacteria. In addition, the ProQ protein has recently been identified as a previously overlooked global RBP in Salmonella enterica (8). As new methods are developed to identify RPBs in bacteria globally, it is essential to be able to analyze their functions systematically to understand their cellular and physiological roles (9).Cold-shock proteins (CSPs) constitute the largest nonribosomal RBP family in Gram-negative bacteria, including the model species Escherichia coli and Salmonella enterica. Their conserved nucleic acid-binding cold-shock domain makes them members of a widespread RBP superfamily that includes the well-investigated eukaryotic Y-box proteins (10, 11). Of the nine and six CSP paralogs present in E. c...

Persisters are nongrowing, transiently antibiotic-tolerant bacteria within a clonal population of otherwise susceptible cells. Their formation is triggered by environmental cues and involves the main bacterial stress response pathways that allow persisters to survive many harsh conditions, including antibiotic exposure. During infection, bacterial pathogens are exposed to a vast array of stresses in the host and form nongrowing persisters that survive both antibiotics and host immune responses, thereby most likely contributing to the relapse of many infections. While antibiotic persisters have been extensively studied over the last decade, the bulk of the work has focused on how these bacteria survive exposure to drugs in vitro. The ability of persisters to survive their interaction with a host is important yet underinvestigated. In order to tackle the problem of persistence of infections that contribute to the worldwide antibiotic resistance crisis, efforts should be made by scientific communities to understand and merge these two fields of research: antibiotic persisters and host-pathogen interactions. Here we give an overview of the history of the field of antibiotic persistence, report evidence for the importance of persisters in infection, and highlight studies that bridge the two areas.

Unlike proteins, RNA molecules have emerged lately as key players in regulation in bacteria. Most reviews hitherto focused on the experimental and/or in silico methods used to identify genes encoding small RNAs (sRNAs) or on the diverse mechanisms of these RNA regulators to modulate expression of their targets. However, less is known about their biological functions and their implications in various physiological responses. This review aims to compile what is known presently about the diverse roles of sRNA transcripts in the regulation of metabolic processes, in different growth conditions, in adaptation to stress and in microbial pathogenesis. Several recent studies revealed that sRNA molecules are implicated in carbon metabolism and transport, amino acid metabolism or metal sensing. Moreover, regulatory RNAs participate in cellular adaptation to environmental changes, e.g. through quorum sensing systems or development of biofilms, and analyses of several sRNAs under various physiological stresses and culture conditions have already been performed. In addition, recent experiments performed with Gram-positive and Gramnegative pathogens showed that regulatory RNAs play important roles in microbial virulence and during infection. The combined results show the diversity of regulation mechanisms and physiological processes in which sRNA molecules are key actors.

Infections by opportunistic bacteria have significant contributions to morbidity and mortality of hospitalized patients and also lead to high expenses in healthcare. In this setting, one of the major clinical problems is caused by Gram-positive bacteria such as enterococci and staphylococci. In this study we extract, purify, identify and characterize immunogenic surface-exposed proteins present in the vancomycin resistant enterococci (VRE) strain Enterococcus faecium E155 using three different extraction methods: trypsin shaving, biotinylation and elution at high pH. Proteomic profiling was carried out by gel-free and gel-nanoLC-MS/MS analyses. The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation. An exclusively extracytoplasmic localization was predicted in 39 (10%) by trypsin shaving, in 47 (15%) by elution at high pH, and 27 (63%) by biotinylation. Comparison between the three extraction methods by Venn diagram and subcellular localization predictors (CELLO v.2.5 and Gpos-mPLoc) allowed us to identify six proteins that are most likely surface-exposed: the SCP-like extracellular protein, a low affinity penicillin-binding protein 5 (PBP5), a basic membrane lipoprotein, a peptidoglycan-binding protein LysM (LysM), a D-alanyl-D-alanine carboxypeptidase (DdcP) and the peptidyl-prolyl cis-trans isomerase (PpiC). Due to their close relationship with the peptidoglycan, we chose PBP5, LysM, DdcP and PpiC to test their potential as vaccine candidates. These putative surface-exposed proteins were overexpressed in Escherichia coli and purified. Rabbit polyclonal antibodies raised against the purified proteins were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Passive immunization with rabbit antibodies raised against these proteins reduced significantly the colony counts of E. faecium E155 in mice, indicating the effectiveness of these surface-related proteins as promising vaccine candidates to target different enterococcal pathogens.

Phylogenetic analysis of the crystal structure of the Enterococcus faecalis SlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator in E. faecalis biology, we dissected the genetic organization of the slyA-EF_3001 locus and constructed a slyA deletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ⌬slyA mutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that the slyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ⌬slyA mutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

f Candidate small RNAs (sRNAs) have recently been identified in Enterococcus faecalis, a Gram-positive opportunistic pathogen, and six of these candidate sRNAs with unknown functions were selected for a functional study. Deletion mutants and complemented strains were constructed, and their virulence was tested. We were unable to obtain the ef0869-0870 mutant, likely due to an essential role, and the ef0820-0821 sRNA seemed not to be involved in virulence. In contrast, the mutant lacking ef0408-0409 sRNA, homologous to the RNAII component of the toxin-antitoxin system, appeared more virulent and more able to colonize mouse organs. The three other mutants showed reduced virulence. In addition, we checked the responses of these mutant strains to several stresses encountered in the gastrointestinal tract or during the infection process. In parallel, the activities of the sRNA promoters were measured using transcriptional fusion constructions. To attempt to identify the regulons of these candidate sRNAs, proteomics profiles of the mutant strains were compared with that of the wild type. This showed that the selected sRNAs controlled the expression of proteins involved in diverse cellular processes and the stress response. The combined data highlight the roles of certain candidate sRNAs in the adaptation of E. faecalis to environmental changes and in the complex transition process from a commensal to a pathogen.

Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen.

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