Streptococcus pneumoniae Rgg/SHP Quorum-sensing Networks and Upstream Regulators of Rgg/SHP1518

In bacteria, quorum-sensing (QS) is a mechanism by which neighboring cells communicate via secreted signaling molecules to regulate cell behaviors. In Streptococcus pneumoniae, seven Rgg/SHP QS systems have been characterized recently. Notably, many of these Rgg/SHP QS systems affect the expression of an operon controlled by the Rgg/SHP1518 QS system. Motivated by the importance of Rgg/SHP1518, I am interested to understand the environmental factors and upstream regulators that activate Rgg/SHP1518 QS. A LacZ reporter driven by the shp1518 promoter regulated by Rgg/SHP1518 was constructed and used with a transposon mutagenesis platform to screen Rgg/SHP1518 QS repressors/inhibitors. Of 110,000 screened transposon insertion mutants, I identified 45 hits to pepO, an annotated endopeptidase, and 35 hits to spxB, a pyruvate oxidase. Given the scale of our transposon screening and the frequency of hits mapped to the two genes, I conclude that pepO and spxB are the only repressors/inhibitors of the Rgg/SHP1518 QS system under the conditions we tested. A hypothetical model for how SpxB regulates Rgg/SHP1518 is proposed. In addition, the mechanism of PepO repression of Rgg/SHP1518 is further illustrated by a set of in vitro experiments. It is demonstrated that pneumococcal PepO requires zinc to degrade SHP1518 and repress Rgg/SHP1518 QS. In addition, the “HExxH” catalytic domain is vital for PepO’s endopeptidase activity. Moreover, the glutamic acid residue in the “HExxH” domain is indispensable for its catalytic function. I expect my work to spark interest and effort in Rgg/SHP QS research, particularly centered on Rgg/SHP1518. I hope the identified inhibitors shed light on studying Rgg/SHP1518 regulation mechanisms.