An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains

Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli

Dual Role for Pilus in Adherence to Epithelial Cells and Biofilm Formation in Streptococcus agalactiae

Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation

SecA localization and SecA-dependent secretion occurs at new division septa in group B Streptococcus.

International audienceExported proteins of Streptococcus agalactiae (GBS), which include proteins localized to the bacterial surface or secreted into the extracellular environment, are key players for commensal and pathogenic interactions in the mammalian host. These proteins are transported across the cytoplasmic membrane via the general SecA secretory pathway and those containing the so-called LPXTG sorting motif are covalently attached to the peptidoglycan by sortase A. How SecA, sortase A, and LPXTG proteins are spatially distributed in GBS is not known. In the close relative Streptococcus pyogenes, it was shown that presence of the YSIRKG/S motif (literally YSIRKX3Gx2S) in the signal peptide (SP) constitutes the targeting information for secretion at the septum. Here, using conventional and deconvolution immunofluorescence analyses, we have studied in GBS strain NEM316 the localization of SecA, SrtA, and the secreted protein Bsp whose signal peptide contains a canonical YSIRKG/S motif (YSLRKykfGlaS). Replacing the SP of Bsp with four other SPs containing or not the YSIRKG/S motif did not alter the localized secretion of Bsp at the equatorial ring. Our results indicate that secretion and cell wall-anchoring machineries are localized at the division septum. Cell wall- anchored proteins displayed polar (PilB, Gbs0791), punctuate (CspA) or uniform distribution (Alp2) on the bacterial surface. De novo secretion of Gbs0791 following trypsin treatment indicates that it is secreted at the septum, then redistributed along the lateral sides, and finally accumulated to the poles. We conclude that the ±YSIRK SP rule driving compartimentalized secretion is not true in S. agalactiae

Distribution of SecA in <i>S.</i><i>agalactiae</i> NEM316.

<p>Bacteria grown overnight in 10 ml of TH (OD₆₀₀≈2) were diluted to get an initial OD₆₀₀ of 0.05 (1/40 dilution) and grown at 37°C until OD₆₀₀ reached 0.5 and re-diluted again in TH (1/10) until reaching an OD₆₀₀ of 0.5 and diluted again (1/10) before final collection at mid-exponential phase (OD₆₀₀ of 0.3) to get a homogenous population of exponentially growing cells. Bacteria were pretreated with lysozyme (1 mg/mL final concentration) for 15 min at 37°C and then permeabilized with PBS-Triton X-100 (0.4%) for 5 min at RT, washed twice with PBS and then fixed with PBS containing 3% paraformaldehyde for 15 min at RT. (A) Differential interference contrast (DIC) and immunofluorescence microscopy (IFM) of bacteria harvested in mid-exponential phase and visualized with rabbit anti-SecA pAb (red) or fluorescent vancomycin (green) plus rabbit anti-SecA pAb (red). White arrows in the last panel indicate potential constriction septa. Image representative of at least 800 GBS chains analyzed (B) Deconvolution images of sequential z-sections (0.3 µm) of NEM316 cells labeled with rabbit anti-SecA pAb presented as maximum intensity projections.</p

Surface distribution of Bsp in <i>S.</i><i>agalactiae</i> NEM316 and derivatives.

<p>Bacteria were harvested in exponential phase (OD₆₀₀ = 0.3) and labeled with DAPI (blue) plus rabbit anti-Bsp pAb (green). The bacterial strains studied were: NEM316 (WT) (positive control); NEM316Δ<i>bsp</i>/pTCV-erm without insert (negative control); NEM316Δ<i>bsp</i>/pTCV-<i>erm</i> expressing recombinant Bsp with the signal peptide of CspA, Alp2, PilB, or Gbs0791. Data are representative of three independent experiments.</p

Poliovirus transcytosis through M-like cells.

During the digestive-tract phase of infection, poliovirus (PV) is found in the oropharynx and the intestine. It has been proposed that PV enters the organism by crossing M cells, which are scattered in the epithelial sheet covering lymphoid follicles of Peyer's patches. However, PV translocation through M cells has never been demonstrated. A model of M-like cells has been previously developed using monolayers of polarized Caco-2 enterocytes cocultured with lymphocytes isolated from Peyer's patches. In this model, lymphoepithelial interactions trigger the appearance of epithelial cells having morphological and functional characteristics of M cells. We have demonstrated efficient, temperature-dependent PV transcytosis in Caco-2 cell monolayers containing M-like cells. This experimental evidence is consistent with M cells serving as gateways allowing PV access to the basal face of enterocytes, the underlying immune follicle cells, and PV transport toward mesenteric lymph nodes

Expression in GBS of Bsp recombinant proteins with structurally unrelated signal peptides.

<p>(A) <i>Bam</i>HI-<i>Not</i>I PCR fragments carrying the ribosome binding site (RBS) and the signal peptides (SP) of 5 SecA-dependent substrates (Bsp, Alp2, Gbs0791, PilB, and CspA) were fused in frame with a <i>Not</i>I-<i>Pst</i>I PCR fragment coding the Bsp protein devoid of its signal peptide (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065832#pone.0065832.s004" target="_blank">Table S1</a>). The resulting <i>Bam</i>HI-<i>Pst</i>I fragments were cloned downstream the constitutive P<i>tetM</i> promoter from the low-copy-number pTCV<i>-erm</i>. The SP and Bsp sequences are indicated in upper-bold italic characters and upper-bold characters, respectively. The boxed RP motif in all proteins corresponded to the translation of the two internal codons of the <i>Not</i>I restriction site (CGGCCG). All but one SP were predicted with SignalP 4.1 (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">www.cbs.dtu.dk/services/SignalP/</a>) whereas the remaining (Alp2) was predicted with PrediSi (<a href="http://www.predisi.de" target="_blank">www.predisi.de</a>). The AA residues in the SP thought to direct localized secretion at the bacterial surface are indicated in red characters. Arrowheads indicate the predicted site of cleavage of the various SP. (B) Analysis of surface display of Bsp recombinant proteins in a Δ<i>bsp</i> mutant strain by immunoblotting. Whole bacterial cells harvested in exponential (OD₆₀₀ 0.3) or stationary (OD₆₀₀ 1.2) phases were washed, resuspended in phosphate buffer saline to similar density and spotted on nitrocellulose. Membranes were hybridized with specific anti-Bsp antibodies or with anti-GBS pAb (loading control). (C) Western blotting analysis of culture supernatants. Proteins were separated on 4–12% gradient Tris-acetate Criterion XT SDS-PAGE gel, then transferred onto a nitrocellulose membrane, and detected by immunoblotting with specific anti-Bsp and anti-CAMP antibodies. In (B) and (C), the Δ<i>bsp</i> mutant strain harboring pTCV-<i>erm</i> (negative control) or pTCV-<i>erm</i> directing synthesis of recombinant Bsp proteins associated with Alp2, Gbs0791, PilB, and CspA signal peptides were used.</p

Distribution of SecA at the surface of <i>S.</i><i>agalactiae</i> (GBS NEM316) and <i>S. pyogenes</i> (GAS M18).

<p>(A) Western blot image showing that the polyclonal antibody directed against <i>E. coli</i> SecA recognizes a band of approximately 90 kDa in both GAS M18 and GBS NEM316 strains. (B) Conventional immunofluorescence microscopy showing the differential distribution of SecA at the surface of GBS NEM316 versus GAS strain M18 collected in exponential and stationary phase of growth. Heterogeneity of SecA distribution was quantified by eye following analysis of randomly selected fields.</p

Capsular polysaccharide of Group B Streptococcus mediates biofilm formation in the presence of human plasma.

International audienceGroup B Streptococcus (GBS) is an asymptomatic colonizer of human mucosal surfaces that is responsible for sepsis and meningitis in neonates. Bacterial persistence and pathogenesis often involves biofilm formation. We previously showed that biofilm formation in medium supplemented with glucose is mediated by the PI-2a pilus. Here, biofilm formation was tested in cell culture medium supplemented with human plasma. GBS strains were able to form biofilms in these conditions unlike Group A Streptococcus (GAS) or Staphylococcus aureus. Analysis of mutants impaired for various surface components revealed that the GBS capsule is a key component in this process

Surface localization of SrtA and unrelated cell wall-anchored proteins in <i>S.</i><i>agalactiae</i> NEM316.

<p>(A, B) Bacteria harvested in exponential phase (OD₆₀₀ = 0.3) were labeled with (A) DAPI (blue) or guinea pig anti-SrtA pAb (green) and (B) with rabbit pAb against PilB, Gbs0791, Alp2, or CspA (red).</p