Investigation of the role of a large serine rich repeat protein in Streptococcus pneumoniae

Streptococcus pneumoniae is a genetically diverse organism that varies substantially in its genomic content from one strain to another. Current therapeutic strategies in the management of pneumococcal disease include treatment with antibiotics and prevention by vaccination. However, due to the highly competent nature of the bacterium the prevalence of antibiotic resistance and vaccine escape is increasing. The pneumococcus causes a wide range of diseases, and this can be attributed to both the succeptibility of the human host and the genetic background of the infecting strain. The study of the contribution of variations in the genome of S.pneumoniae is clearly important in understanding the behaviour of this organism, and managing the burden of disease relating to this organism. S.pneumoniae strains are able to acquire DNA from other strains, and also from other closely related species, who occupy the same niche in the human host. One region of genomic diversity in the pneumococcus encodes a large serine rich repeat protein, glycoysltransferases and secretion proteins, some of which are homologous to the Sec secretion pathway. Similar loci have been characterised and found to be important in the virulence of other gram positive bacteria, including S.gordonii and S.parasanguinis. The presence of this locus was investigated in a diverse population of pneumococcal isolates, and shown to be present in a wide variety of isolates. The RNA of genes in the locus was found to be expressed. Expression of the SRR protein, encoded by SP1772, was investigated; a role in biofilm formation was identified utilising an isogenic mutant in SP1772 of TIGR4. In addition, the gene encoding the SRR was found to be able to recombine within a single strain of S.pneumoniae, suggesting this region of the genome is not only variable in its presence in the pneumococcal population but also able to adapt to the environment it is in

Transcriptional organization of pneumococcal psrP-secY2A2 and impact of GtfA and GtfB deletion on PsrP-associated virulence properties

Pneumococcal serine-rich repeat protein (PsrP) is a glycoprotein that mediates Streptococcus pneumoniae attachment to lung cells and promotes biofilm formation. Herein, we investigated the transcriptional organization of psrP-secY2A2, the 37-kbp pathogenicity island encoding PsrP and its accessory genes. PCR amplification of cDNA and RNA-seq analysis found psrP-secY2A2 to be minimally composed of three operons: psrP-glyA, glyB, and glyC-asp5. Transcription of all three operons was greatest during biofilm growth and immunoblot analyses confirmed increased PsrP production by biofilm pneumococci. Using gas chromatography-mass spectrometry we identified monomeric N-acetylglucosamine as the primary glycoconjugate present on a recombinant intracellular version of PsrP, i.e. PsrP1-734. This finding was validated by immunoblot using lectins with known carbohydrate specificities. We subsequently deleted gtfA and gtfB, the GTFs thought to be responsible for addition of O-linked N-acetylglucosamine, and tested for PsrP and its associated virulence properties. These deletions negatively affected our ability to detect PsrP1-734 in bacterial whole cell lysates. Moreover, S. pneumoniae mutants lacking these genes pheno-copied the psrP mutant and were attenuated for: biofilm formation, adhesion to lung epithelial cells, and pneumonia in mice. Our studies identify the transcriptional organization of psrP-secY2A2 and show the indispensable role of GtfA and GtfB on PsrP-mediated pneumococcal virulence