RrgA is a pilus-associated adhesin in Streptococcus pneumoniae

Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production

The XMM-Newton serendipitous ultraviolet source survey catalogue

The XMM-Newton Serendipitous Ultraviolet Source Survey (XMM-SUSS) is a catalogue of ultraviolet (UV) sources detected serendipitously by the Optical Monitor (XMM-OM) on-board the XMM-Newton observatory. The catalogue contains ultraviolet-detected sources collected from 2,417 XMM-OM observations in 1-6 broad band UV and optical filters, made between 24 February 2000 and 29 March 2007. The primary contents of the catalogue are source positions, magnitudes and fluxes in 1 to 6 passbands, and these are accompanied by profile diagnostics and variability statistics. The XMM-SUSS is populated by 753,578 UV source detections above a 3 sigma signal-to-noise threshold limit which relate to 624,049 unique objects. Taking account of substantial overlaps between observations, the net sky area covered is 29-54 square degrees, depending on UV filter. The magnitude distributions peak at 20.2, 20.9 and 21.2 in UVW2, UVM2 and UVW1 respectively. More than 10 per cent of sources have been visited more than once using the same filter during XMM-Newton operation, and > 20 per cent of sources are observed more than once per filter during an individual visit. Consequently, the scope for science based on temporal source variability on timescales of hours to years is broad. By comparison with other astrophysical catalogues we test the accuracy of the source measurements and define the nature of the serendipitous UV XMM-OM source sample. The distributions of source colours in the UV and optical filters are shown together with the expected loci of stars and galaxies, and indicate that sources which are detected in multiple UV bands are predominantly star-forming galaxies and stars of type G or earlier.Comment: Accepted for publication in MNRA

Cloning, expression, purification, crystallization and preliminary X-ray analysis of the pilus-associated sortase C from Streptococcus pneumoniae

Crystallization conditions and preliminary X-ray diffraction analysis of the S. pneumoniae-derived pilus-associated protein sortase C are reported

Recombinant RrgA and RrgC proteins bind to human respiratory epithelial cells

<p><b>Copyright information:</b></p><p>Taken from "RrgA is a pilus-associated adhesin in "</p><p></p><p>Molecular Microbiology 2007;66(2):329-340.</p><p>Published online Jan 2007</p><p>PMCID:PMC2170534.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> A–C. Recombinant RrgA binds directly to A549 respiratory epithelial cells. Cells were incubated with 100 μg ml of purified RrgA (A1 and A2, ‘+RrgA’) or GFP (B1 and B2, ‘+GFP’) in DMEM culture medium, or medium alone (A3–4, ‘-RrgA’; B3–4, ‘-GFP’) for 2 h at 4°C. Cells were fixed and stained with anti-RrgA and anti-GFP antibodies (A1–4, ‘αRrgA’; B1–4, ‘αGFP’) and phalloidin, which served as a control to demonstrate the presence of cells (A2, A4, B2 and B4). Imaging was performed with a confocal microscope. Scale bar is 20 μm. C. RrgA and RrgC bind to A549 cells in a dose-dependent manner. A549 cells were mixed in suspension with either medium alone (0 μg ml) or three concentrations (5, 50 and 100 μg ml) of pilus subunits RrgA (squares with solid black lines), RrgB (triangles with dashed grey lines), RrgC (upside-down triangles with dashed black lines), or GFP protein (diamond with solid grey lines), incubated for 2 h at 4°C, stained with antisera specific to each protein, and detected with Alexa Fluor 488-conjugated secondaries. Cells were analysed with a FACS-Calibur flow cytometer, and the net mean fluorescence intensity for each population was calculated from three independent experiments. Significant differences were detected by repeated-measure (< 0.0001), and both RrgA and RrgC binding was significantly different from RrgB and GFP binding at 50 and 100 μg ml by Bonferroni analysis (* < 0.001). D–G. Pre-incubation of A549 cells with purified RrgA protein inhibits pilus-mediated adherence. A549 cells were pre-incubated with media alone (D), media containing 100 μg ml of RrgA (E), or 100 μg ml of GFP (F). After pre-incubation, A549 monolayers were infected with strain T4. Cells were stained with phalloidin (red) anti- capsule antibody (green), and imaged with a confocal microscope. RrgA pre-incubation inhibits the adherence of strain T4 to the A549 cells (E versus D), while the negative control GFP protein does not (F versus D). Scale bar is 20 μm. Adherent bacteria were counted, and the number of bacteria adherent to 100 A549 cells is shown in G ( = 6 fields, *= 0.0002, **= 0.8)

Expression of confers pilus-mediated adherence to human respiratory epithelial cells

<p><b>Copyright information:</b></p><p>Taken from "RrgA is a pilus-associated adhesin in "</p><p></p><p>Molecular Microbiology 2007;66(2):329-340.</p><p>Published online Jan 2007</p><p>PMCID:PMC2170534.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> A. , but not and , is necessary for pilus-mediated adherence to epithelial cells. Adherence of wild-type piliated TIGR4 (‘T4’) to A549 human respiratory epithelial cells was significantly greater than both non-piliated T4Δ() compared with T4 deficient in the gene (‘T4Δ). Adherence of T4 deficient in both and (‘T4ΔC’) was not notably different from wild-type organisms, but was significantly greater compared with T4Δ. Repeated-measure of data collected from three independent determinations indicates statistically significant differences within experimental conditions. Bonferroni analyses identify specific significant differences: **< 0.01. B. expression determines adherence to epithelial cells. T4 deficient in are significantly deficient in adherence (‘Δ), compared with wild-type (T4), while -complementation restores wild-type adherence [‘Δ∇(::’)]. Introduction of a second copy of inserted in the locus [‘∇(::’] results in significant enhancement of adherence over wild-type levels. Statistical analyses were performed with repeated-measure of data collected from three independent determinations. Bonferroni analyses identify specific significant differences: *< 0.01 and **< 0.001

RrgA is involved in pilus-mediated colonization of the upper respiratory tract in mice

<p><b>Copyright information:</b></p><p>Taken from "RrgA is a pilus-associated adhesin in "</p><p></p><p>Molecular Microbiology 2007;66(2):329-340.</p><p>Published online Jan 2007</p><p>PMCID:PMC2170534.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> Mice were challenged intranasally with a low dose of T4, T4Δ or T4Δ (. 7 × 10 cfu per mouse). Bacterial density in the nasopharynx was determined 7 days post infection. Mice infected with T4Δ had significantly fewer bacteria in the nasopharynx than mice infected with T4 or T4Δ (*< 0.01, = 3 independent replicates of 10 mice per group per replicate, using the Kruskal–Wallis test with Dunn's post testing)

Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 36FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.Fil: Sarnacki, Sebastian Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina;Fil: Aya Castañeda, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina;Fil: Noto Llana, Mariangeles. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina;Fil: Giacomodonato, Mónica Nancy. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina;Fil: Valvano, Miguel Angel. The Queens University Of Belfast;Fil: Cerquetti, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina