Secretion, Maturation, and Activity of a Quorum Sensing Peptide (GSP) Inducing Bacteriocin Transcription in Streptococcus gallolyticus.

subsp. is an emerging opportunistic pathogen responsible for septicemia and endocarditis in the elderly. Invasive infections by subsp. are strongly linked to the occurrence of colorectal cancer (CRC). It was previously shown that increased secondary bile salts under CRC conditions enhance the bactericidal activity of gallocin, a bacteriocin produced by subsp. , enabling it to colonize the mouse colon by outcompeting resident enterococci (L. Aymeric, F. Donnadieu, C. Mulet, L. du Merle, et al., Proc Natl Acad Sci U S A 115:E283-E291, 2018, https://doi.org/10.1073/pnas.1715112115). In a separate study, we showed that subsp. produces and secretes a 21-mer peptide that activates bacteriocin production (A. Proutière, L. du Merle, B. Périchon, H. Varet, et al., mBio 11:e03187-20, 2020, https://doi.org/10.1128/mBio.03187-20). This peptide was named CSP because of its sequence similarity with competence-stimulating peptides found in other streptococci. Here, we demonstrate that CSP is a bona fide quorum sensing peptide involved in activation of gallocin gene transcription. We therefore refer to CSP as GSP (gallocin-stimulating peptide). GSP displays some unique features, since its N-terminal amino acid lies three residues after the double glycine leader sequence. Here, we set out to investigate the processing and export pathway that leads to mature GSP. Heterologous expression in of the genes encoding GSP and the BlpAB transporter is sufficient to produce the 21-mer form of GSP in the supernatant, indicating that subsp. BlpAB displays an atypical cleavage site. We also conducted the first comprehensive structure-activity relationship (SAR) analysis of subsp. GSP to identify its key structural features and found that unlike many other similar streptococci signaling peptides (such as CSPs), nearly half of the mature GSP sequence can be removed (residues 1 to 9) without significantly impacting the peptide activity. subsp. is an opportunistic pathogen associated with colorectal cancer (CRC) and endocarditis. subsp. utilizes quorum sensing (QS) to regulate the production of a bacteriocin (gallocin) and gain a selective advantage in colonizing the colon. In this article, we report (i) the first structure-activity relationship study of the subsp. QS pheromone that regulates gallocin production, (ii) evidence that the active QS pheromone is processed to its mature form by a unique ABC transporter and not processed by an extracellular protease, and (iii) supporting evidence of interspecies interactions between streptococcal pheromones. Our results revealed the minimal pheromone scaffold needed for gallocin activation and uncovered unique interactions between two streptococcal QS signals that warrant further study.