Identification and Characterization of Negative Regulators of Rgg1518 Quorum Sensing in Streptococcus pneumoniae

Streptococcus pneumoniae uses quorum sensing to communicate and regulate virulence. In our study, we focused on one Rgg quorum sensing system (Rgg/SHP1518) and found that multiple other Rgg regulators also control it. Streptococcus pneumoniae is an agent of otitis media, septicemia, and meningitis and remains the leading cause of community-acquired pneumonia regardless of vaccine use. Of the various strategies that S. pneumoniae takes to enhance its potential to colonize the human host, quorum sensing (QS) is an intercellular communication process that provides coordination of gene expression at a community level. Numerous putative QS systems are identifiable in the S. pneumoniae genome, but their gene-regulatory activities and contributions to fitness have yet to be fully evaluated. To contribute to assessing regulatory activities of rgg paralogs present in the D39 genome, we conducted transcriptomic analysis of mutants of six QS regulators. Our results find evidence that at least four QS regulators impact the expression of a polycistronic operon (encompassing genes spd_1517 to spd_1513) that is directly controlled by the Rgg/SHP1518 QS system. As an approach to unravel the convergent regulation placed on the spd_1513-1517 operon, we deployed transposon mutagenesis screening in search of upstream regulators of the Rgg/SHP1518 QS system. The screen identified two types of insertion mutants that result in increased activity of Rgg1518-dependent transcription, one type being where the transposon inserted into pepO, an annotated endopeptidase, and the other type being insertions in spxB, a pyruvate oxidase. We demonstrate that pneumococcal PepO degrades SHP1518 to prevent activation of Rgg/SHP1518 QS. Moreover, the glutamic acid residue in the conserved "HExxH" domain is indispensable for the catalytic function of PepO. Finally, we confirmed the metalloendopeptidase property of PepO, which requires zinc ions, but not other ions, to facilitate peptidyl hydrolysis.IMPORTANCE Streptococcus pneumoniae uses quorum sensing to communicate and regulate virulence. In our study, we focused on one Rgg quorum sensing system (Rgg/SHP1518) and found that multiple other Rgg regulators also control it. We further identified two enzymes that inhibit Rgg/SHP1518 signaling and revealed and validated one enzyme's mechanisms for breaking down quorum sensing signaling molecules. Our findings shed light on the complex regulatory network of quorum sensing in Streptococcus pneumoniae.