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Streptococcus pneumoniae is able to integrate exogenous DNA into its genome by natural genetic transformation. Transient accumulation of high levels of the only S. pneumoniae alternative factor is insufficient for development of full competence without expression of a second competence-specific protein, ComW. The ⌬comW mutant is 10 4 -fold deficient in the yield of recombinants, 10-fold deficient in the amount of X activity, and 10-fold deficient in the amount of X protein. The critical role of ComW during transformation can be partially obviated by A mutations clustered on surfaces controlling affinity for core RNA polymerase (RNAP). While strains harboring A mutations in the comW mutant background were transforming at higher rates, the mechanism of transformation restoration was not clear. To investigate the mechanism of transformation restoration, we measured late gene expression in A * suppressor strains. Restoration of late gene expression was observed in ⌬comW A * mutants, indicating that a consequence of the A * mutations is, at least, to restore X activity. Competence kinetics were normal in ⌬comW A * strains, indicating that strains with restored competence exhibit the same pattern of transience as wild-type (WT) strains. We also identified a direct interaction between ComW and X using the yeast two-hybrid (Y2H) assay. Taken together, these data are consistent with the idea that ComW increases X access to core RNAP, pointing to a direct role of ComW in factor exchange during genetic transformation. However, the lack of late gene shutoff in ⌬comW mutants also points to a potential new role for ComW in competence shutoff. IMPORTANCEThe sole alternative sigma factor of the streptococci, SigX, regulates development of competence for genetic transformation, a widespread mechanism of adaptation by horizontal gene transfer in this genus. The transient appearance of this sigma factor is strictly controlled at the levels of transcription and stability. This report shows that it is also controlled at the point of its substitution for SigA by a second transient competence-specific protein, ComW. Streptococcus pneumoniae, a Gram-positive opportunistic pathogen found in the human nasopharynx, causes diseases such as pneumonia and meningitis. S. pneumoniae's natural competence, or ability to integrate exogenous DNA into its chromosome, provides a major mechanism of rapidly overcoming selective pressure (1, 2). The ability to take up and exchange DNA depends on development of the competent state, which is prompted by quorum sensing (QS) mediated by a small peptide, via a signal transduction pathway that is incompletely understood.Cells in the exponential growth phase are first primed for transformation by a QS mechanism encoded by two genetic loci initially transcribed at a basal level by core RNA polymerase (RNAP) bound to the primary sigma factor, A (3). The loci are comAB (4, 5) and comCDE (3). ComC is a propeptide cleaved and exported by ComAB, as the mature peptide called CSP (competence-stimulating pepti...

Background Polyps from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) contain increased levels of autoreactive antibodies, B cells and fibrin deposition. Anti‐phospholipid antibodies (APA) are autoantibodies known to cause thrombosis but have not been implicated in chronic rhinosinusitis (CRS). Objective To compare APA levels (anti‐cardiolipin, anti‐phosphatidylethanolamine (anti‐PE), and anti‐β2‐glycoprotein (anti‐B2GP)) in nasal polyp (NP) tissue with tissue from control and CRS without nasal polyp (CRSsNP) patients, we tested whether NP antibodies affect coagulation, and correlate APAs with anti‐dsDNA IgG and markers of coagulation. Methods Patient specimens were assayed for APA IgG, anti‐dsDNA IgG and thrombin‐anti‐thrombin (TaT) complex by ELISA. Antibodies from a subset of specimens were tested for modified activated partial thromboplastin time (aPTT) measured on an optical‐mechanical coagulometer. Results Anti‐cardiolipin IgG in NP was 5‐fold higher than control tissue (p < .0001). NP antibodies prolonged aPTT compared to control tissue antibodies at 400 µg/mL (36.7 s vs. 33.8 s, p = .024) and 600 µg/mL (40.9 s vs. 34.7 s, p = .0037). Anti‐PE IgG antibodies were increased in NP (p = .027), but anti‐B2GP IgG was not significantly higher (p = .084). All APAs correlated with anti‐dsDNA IgG levels, which were also elevated (R = .77, .71 and .54, respectively, for anti‐cardiolipin, anti‐PE, and anti‐B2GP; all p < .001), but only anti‐cardiolipin (R = .50, p = .0185) and anti‐PE (R = 0.45, p = .037) correlated with TaT complex levels. Conclusions APA IgG antibodies are increased in NP and correlate with autoreactive tissue antibodies. NP antibodies have in vitro anti‐coagulant activity similar to those observed in anti‐phospholipid syndrome, suggesting that they may have pro‐coagulant effects in polyp tissue.

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Background We have previously found that anti-dsDNA IgG autoantibodies are increased in Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) patients. Recent studies find that B cells with the Epstein-Barr virus-induced protein 2(EBI2), is critical to the extrafollicular response. We have described elevated numbers of EBI2+ B cells in NP and they were more frequently antibody secreting cells (ASCs). In this study we evaluate whether dsDNA-specific ASCs are increased in NP and further compared the EBI2+ and EBI2− compartments. Methods To investigate this problem, flow cytometry and ELISpot assays were utilized to compare the frequency of total IgG and dsDNA specific IgG ASCs and cells were sorted into EBI2+ and EBI2− B cells from human NP and tonsils. Results Flow cytometry results (n=10 tonsil, 4 NP) found that although tonsils had significantly more B cells than NP, a significantly smaller proportion were CD27+CD38+ plasmablasts (PB). Further ELISpot analysis found NP B cells were more frequently IgG ASCs than tonsil (0.06% and 0.43% in tonsil (n=9) and NP (n=5) respectively, p&lt;0.001). Anti-dsDNA specific ASCs were two-fold more in NP representing 0.95% of IgG ASCs compared with 0.4% in tonsil ASCs (p&lt;0.05). In analysis of ASCs sorted from tonsils (n=3) and NP (n=2), we found anti-dsDNA IgG ASCs were more frequent among EBI2+ compared to EBI2− cells with 10- and 14-fold increases in dsDNA reactivity (p&lt;0.01). Conclusions Although B-cells are significantly more abundant in tonsils, NP derived B-cells more frequently secrete total- and dsDNA-specific IgG. These differences appear to be driven by extrafollicular EBI2+ B-cells suggesting that EBI2+ B cells represent the major pool of autoreactive B-cells observed in CRSwNP and tonsils.