The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

SummaryStaphylococcus aureus biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA/PNAG), the product of the ica operon. The staphylococcal accessory regulator, SarA, is a central regulatory element that controls the production of S. aureus virulence factors. By screening a library of Tn917 insertions in a clinical S. aureus strain, we identified SarA as being essential for biofilm development. Non-polar mutations of sar A in four genetically unrelated S. aureus strains decreased PIA/PNAG production and completely impaired biofilm development, both in steady state and flow conditions via an agr -independent mechanism. Accordingly, real-time PCR showed that the mutation in the sar A gene resulted in downregulation of the ica operon transcription. We also demonstrated that complete deletion of s s s s B did not affect PIA/PNAG production and biofilm formation, although it slightly decreased ica operon transcription. Furthermore, the sar A-s s s s B double mutant showed a significant decrease of ica expression but an increase of PIA/PNAG production and biofilm formation compared to the sar A single mutant. We propose that SarA activates S. aureus development of biofilm by both enhancing the ica operon transcription and suppressing the transcription of either a protein involved in the turnover of PIA/PNAG or a repressor of its synthesis, whose expression would be s s s s B

SummaryIn environmental settings, biofilms represent the common way of life of microorganisms. Salmonella enterica serovar Enteritidis, the most frequent cause of gastroenteritis in developed countries, produces a biofilm whose matrix is mainly composed of curli fimbriae and cellulose. In contrast to other bacterial biofilms, no proteinaceous compound has been reported to participate in the formation of this matrix. Here, we report the discovery of BapA, a large cellsurface protein required for biofilm formation by S. Enteritidis. Deletion of bap A caused the loss of the capacity to form a biofilm whereas the overexpression of a chromosomal copy of bap A increased the biofilm biomass formation. We provide evidence that overproduction of curli fimbriae and not cellulose can compensate for the biofilm deficiency of a bap A mutant strain. BapA is secreted through a type I protein secretion system (BapBCD) situated downstream of the bap A gene and was found to be loosely associated with the cell surface. Experiments with mixed bacterial populations positive or negative for BapA showed that BapA minus cells are not recruited into the biofilm matrix. The expression of bapA is coordinated with that of genes encoding curli fimbriae and cellulose, through the action of csg D. Studies on the contribution of BapA to S. Enteritidis pathogenesis revealed that orally inoculated animals with a bap Adeficient strain survived longer than those inoculated with the wild-type strain. Also, a bap A mutant strain showed a significantly lower colonization rate at the intestinal cell barrier and consequently a decreased efficiency for organ invasion compared with the wildtype strain. Taken together, these data demonstrate that BapA contributes both to biofilm formation and invasion through the regular Salmonella infection route.

Infection by the bacterium Listeria monocytogenes depends on host cell clathrin. To determine whether this requirement is widespread, we analyzed infection models using diverse bacteria. We demonstrated that bacteria that enter cells following binding to cellular receptors (termed "zippering" bacteria) invade in a clathrin-dependent manner. In contrast, bacteria that inject effector proteins into host cells in order to gain entry (termed "triggering" bacteria) invade in a clathrin-independent manner. Strikingly, enteropathogenic Escherichia coli (EPEC) required clathrin to form actin-rich pedestals in host cells beneath adhering bacteria, even though this pathogen remains extracellular. Furthermore, clathrin accumulation preceded the actin rearrangements necessary for Listeria entry. These data provide evidence for a clathrin-based entry pathway allowing internalization of large objects (bacteria and ligand-coated beads) and used by "zippering" bacteria as part of a general mechanism to invade host mammalian cells. We also revealed a nonendocytic role for clathrin required for extracellular EPEC infections.

RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5′ and 3′ untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels.antisense RNA | overlapping transcription | RNA processing | posttranscriptional regulation | microRNA F or many years, the catalog of transcripts (transcriptome) produced by bacterial cells was limited to the transcription products of known annotated genes (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). In the past 10 years, the development of new approaches based on high-resolution tiling arrays and RNA deep sequencing (RNA-seq) has uncovered that a significant proportion (depending on the study, varying between 3% and >50%) of protein coding genes are also transcribed from the reverse complementary strand (1-17). In most cases, overlapping transcription generates a noncoding antisense transcript whose size can vary between various tens of nucleotides (cisencoded small RNAs) to thousands of nucleotides (antisense RNAs). The antisense transcript can cover the 5′ end, 3′ end, middle, entire gene, or even various contiguous genes. Alternatively, overlapping transcription can also be due to the overlap between long 5′ or 3′ UTRs of mRNAs transcribed in the opposite direction. Independent of the mechanism by which it is generated, overlapping transcription has been proposed to affect the expression of the target gene at different levels [for review, see Thomason and Storz (18)]. These mechanisms include: (i) the overlapped transcript affects the stability of the target RNA by either promoting (RNA degradation) or blocking (RNA stabilization) cleavage by endoribonucleases or exoribonucleases; (ii) the overlapped transcript induces a change in the structure of the mRNA that affects transcription termination (transcription attenuation); (iii) the overlapped transcript prevents RNA polymerase from binding or extending the transcript encoded in the opposite strand (transcription interference); and (iv) the overl...

The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.Pathogenic bacteria often interact with their host through surface proteins referred to as adhesins. Two major classes of adhesins have been described, and both utilize vastly different mechanisms for their assembly at the cell surface. Fimbrial adhesins such as the prototypical type 1 fimbriae of Escherichia coli are composed primarily of a major repeating subunit protein and often contain minor-subunit proteins (including the adhesin) at the tip of the organelle. The biogenesis of these fimbriae is dependent on a highly conserved chaperone-usher system (20,29). Nonfimbrial adhesins are a second class of adherence factors and encompass a diverse range of adhesins that includes the autotransporter (AT) family of proteins. AT proteins are particularly unique in that the information required for receptor recognition and routing and anchorage to the outer membrane is provided by the protein itself (25). Recent studies have identified a new group of nonfimbrial adhesins that have the capacity to form stable trimeric structures on the bacterial cell surface. These adhesins are characterized by a membrane-anchored C-terminal domain that forms a trimeric ␤-barrel pore and facilitates the translocation of a passenger domain (consisting of an extended stalk and an N-terminal head) to the bacterial cell surface via the type V secretion pathway (54, 67).Trimeric AT proteins have been identified from a range of different gram-negative bacterial pathogens. Where characterized, the function of trimeric AT proteins is universally associated with bacterial adherence, and thus they constitute an important group of virulence factors (14). The YadA adhesin from Yersinia enterocolitica represents the best-characterized trimeric AT protein. YadA mediates adherence to host epithelial cells an...