Project description:Infection of the human host by Streptococcus pneumoniae begins with colonization of the nasopharynx, which is mediated by adherence of bacteria to respiratory epithelium. Several studies have indicated an important role for the pneumococcal capsule in this process. Here, we used microarrays to characterize the in vitro transcriptional response of human nasopharyngeal epithelial Detroit 562 cells to adherence of serotype 2-encapsulated strain D39, serotype 19F-encapsulated strain G54, serotype 4-encapsulated strain TIGR4, and their nonencapsulated derivatives (delta-cps). In total, 322 genes were found to be upregulated in response to adherent pneumococci. Twenty-two genes were commonly induced, including those encoding several cytokines (e.g., IL-1-beta, IL-6), chemokines (e.g., IL-8, CXCL1/2), and transcriptional regulators (e.g., FOS), consistent with an innate immune response mediated by Toll-like receptor signaling. Interestingly, 85% of genes was induced specifically by one or more encapsulated strains, suggestive of a capsule-dependent response. Importantly, purified capsular polysaccharides alone had no effect. Over a third of these loci encoded products predicted to be involved in transcriptional regulation and signal transduction, in particular MAPK signaling pathways. Real-time PCR of a subset of ten genes confirmed microarray data and showed a time-dependent upregulation of especially innate immunity genes. Downregulation of epithelial genes was most pronounced upon adherent D39delta-cps, as 68% of the 161 genes identified was only repressed using this nonencapsulated strain. In conclusion, we identified a subset of host genes specifically induced by encapsulated strains during in vitro adherence, and have demonstrated the complexity of interactions occurring during the initial stages of pneumococcal infection. Experiment Overall Design: We used three different pneumococcal strains and their isogenic nonencapsulated derivatives (delta-cps): serotype 2-encapsulated strain D39, serotype 19F-encapsulated strain G54, and serotype 4-encapsulated strain TIGR4. All experiments were performed in triplicate (3 independent biological replicates) and compared to uninfected control Detroit 562 cells. Bacteria were allowed to adhere to the epithelial cells for 2 hours, after which the transcriptional response of the Detroit cells was analyzed using Affymetrix Human U133 Plus GeneChips. In addition to the 6 strains mentioned above, we included transcriptional analysis of the epithelial cell response to low-dose D39delta-cps (giving adherence equivalent to wild-type D39) and purified type 2 capsular polysaccharides.

Project description:For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus.

Project description:For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus. Pneumococcal biofilms compared to planktonic control at 4, 12, 24, 48 hours. 3 biological replicates each of 4 and 12 hour time points, and 2 biological replicates each of 24 and 48 hour time points. Flip dye (technical replicates) performed for 4, 12, and 24 hour time points; no technical replicate performed for 48 hour time point due to limiting material. Ratios were determined by averaging across technical and biological replicates. The following hybridizations made up each biological replicate: 14090167.tav.annot and 14090190.tav.annot (4hr biol rep 1); 14090169.tav.annot and 14090176.tav.annot (4hr biol rep 2); 14090192.tav.annot and 14090188.tav.annot (4hr biol rep 3); 14087688.tav.annot and 14090180.tav.annot (12hr biol rep 1); 14090185.tav.annot and 14090168.tav.annot (12hr biol rep 2); 14090191.tav.annot and 14090174.tav.annot (12hr biol rep 3); 14090170.tav.annot and 14090175.tav.annot (24hr biol rep 2); 14090193.tav.annot and 14087687.tav.annot (24hr biol rep 3); 14090181.tav.annot (48hr biol rep 1); 14090187.tav.annot (48hr biol rep 2)

Dataset Information

CGH characterisation of non-typeable Streptococcus pneumococcus circulating in Portugal

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