Flavin Reductase Contributes to Pneumococcal Virulence by Protecting from Oxidative Stress and Mediating Adhesion and Elicits Protection Against Pneumococcal Challenge.

Pneumococcal flavin reductase (FlaR) is known to be cell-wall associated and possess age dependent antigenicity in children. This study aimed at characterizing FlaR and elucidating its involvement in pneumococcal physiology and virulence. Bioinformatic analysis of FlaR sequence identified three-conserved cysteine residues, suggesting a transition metal-binding capacity. Recombinant FlaR (rFlaR) bound Fe2+ and exhibited FAD-dependent NADP-reductase activity, which increased in the presence of cysteine or excess Fe2+ and inhibited by divalent-chelating agents. flaR mutant was highly susceptible to H2O2 compared to its wild type (WT) and complemented strains, suggesting a role for FlaR in pneumococcal oxidative stress resistance. Additionally, flaR mutant demonstrated significantly decreased mice mortality following intraperitoneal infection. Interestingly, lack of FlaR did not affect the extent of phagocytosis by primary mouse peritoneal macrophages but reduced adhesion to A549 cells compared to the WT and complemented strains. Noteworthy are the findings that immunization with rFlaR elicited protection in mice against intraperitoneal lethal challenge and anti-FlaR antisera neutralized bacterial virulence. Taken together, FlaR's roles in pneumococcal physiology and virulence, combined with its lack of significant homology to human proteins, point towards rFlaR as a vaccine candidate.

Exit from competence for genetic transformation in Streptococcus pneumoniae is regulated at multiple levels.

Development of natural competence in S. pneumoniae entails coordinated expression of two sets of genes. Early gene expression depends on ComE, a response regulator activated by the pheromone CSP (Competence-Stimulating-Peptide). Subsequently, an early gene product (the alternative sigma factor ComX) activates expression of late genes, establishing the competent state. Expression of both sets of genes is transient, rapidly shut off by a mechanism that depends on the late gene, dprA. It has been thought that the rapid shutoff of late gene expression is the combined result of auto-inhibition of ComE and the instability of ComX. However, this explanation seems incomplete, because of evidence for ComX-dependent repressor(s) that might also be important for shutting off the response to CSP and identifying dprA as such a gene. We screened individual late gene mutants to investigate further the roles of ComX-dependent genes in competence termination. A ΔdprA mutant displayed a prolonged late gene expression pattern, whereas mutants lacking cbpD, cibABC, cglEFG, coiA, ssbB, celAB, cclA, cglABCD, cflAB, or radA, exhibited a wild-type temporal expression pattern. Thus, no other gene than dprA was found to be involved in shutoff. DprA limits the amounts of ComX and another early gene product, ComW, by restriction of early gene expression rather than by promoting proteolysis. To ask if DprA also affects late gene expression, we decoupled late gene expression from early gene regulation. Because DprA did not limit ComX activity under these conditions, we also conclude that ComX activity is limited by another mechanism not involving DprA.

spr1630 is responsible for the lethality of clpX mutations in Streptococcus pneumoniae.

The Clp protease ATPase subunit and chaperone ClpX is dispensable in some bacteria, but it is thought to be essential in others, including streptococci and lactococci. We confirm that clpX is essential in the Rx strain of Streptococcus pneumoniae but show that the requirement for clpX can be relieved by point mutations, frame shifts, or deletion of the gene spr1630, which is found in many isolates of S. pneumoniae. Homologs occur frequently in Staphylococcus aureus as well as in a few strains of Listeria monocytogenes, Lactobacillus johnsonii, and Lactobacillus rhamnosus. In each case, the spr1630 homolog is accompanied by a putative transcriptional regulator with an HTH DNA binding motif. In S. pneumoniae, the spr1630-spr1629 gene pair, accompanied by a RUP element, occurs as an island inserted between the trpA and cclA genes in 15 of 22 sequenced genomes.

Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.

Competence for genetic transformation in Streptococcus pneumoniae is a transient physiological state whose development is coordinated by a peptide pheromone (CSP) and its receptor, which activates transcription of two downstream genes, comX and comW, and 15 other "early" genes. ComX, a transient alternative sigma factor, drives transcription of "late" genes, many of which are essential for transformation. In vivo, ComW both stabilizes ComX against proteolysis by the ClpE-ClpP protease and stimulates its activity. Interestingly, stabilization of ComX by deletion of the gene encoding the ClpP protease did not extend the period of competence. We considered the hypothesis that the rapid decay of competence arises from a rapid loss of ComW and thus of its ComX stimulating activity, so that ComX might persist but lose its transcriptional activity. Western analysis revealed that ComW is indeed a transient protein, which is also stabilized by deletion of the gene encoding the ClpP protease. However, stabilizing both ComX and ComW did not prolong either ComX activity or the period of transformation, indicating that termination of the transcriptional activity of ComX is not dependent on proteolysis of ComW.