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

Abstract

Streptococcus gallolyticus subsp. gallolyticus is an emerging opportunistic pathogen responsible for septicemia and endocarditis in the elderly. Invasive infections by S. gallolyticus subsp. gallolyticus 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 S. gallolyticus subsp. gallolyticus, 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 S. gallolyticus subsp. gallolyticus 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 Lactococcus lactis of the genes encoding GSP and the BlpAB transporter is sufficient to produce the 21-mer form of GSP in the supernatant, indicating that S. gallolyticus subsp. gallolyticus BlpAB displays an atypical cleavage site. We also conducted the first comprehensive structure-activity relationship (SAR) analysis of S. gallolyticus subsp. gallolyticus 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.IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus is an opportunistic pathogen associated with colorectal cancer (CRC) and endocarditis. S. gallolyticus subsp. gallolyticus 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 S. gallolyticus subsp. gallolyticus 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.

Keywords: Streptococcus; bacteriocins; peptide; quorum sensing.

FIG 1

The ABC transporter BlpAB secretes both GSP and gallocin peptides. (A) Summary table of BLAST results comparing the ComABCDE system and SepM proteins of S. mutans with their homologous counterparts in S. gallolyticus subsp. gallolyticus. (B) Agar diffusion assay showing gallocin activity in the culture supernatant against S. gallolyticus subsp. macedonicus. Strains tested include S. gallolyticus subsp. gallolyticus UCN34 ΔblpAB and UCN34 ΔsepM, their bWT counterparts, and complemented S. gallolyticus subsp. gallolyticus UCN34 ΔblpAB containing the plasmid pTCVΩPtetO-blpAB with or without induction of PtetO. (C) Mean fluorescence of the reporter strain S. gallolyticus subsp. gallolyticus UCN34 Δgsp pTCVΩPgllA-gfp resuspended in the supernatant of S. gallolyticus subsp. gallolyticus UCN34 WT, Δgsp, ΔblpAB, bWT blpAB, ΔsepM, and bWT sepM strains. Results are means and standard deviations (SD) from three independent experiments. (D) PgllA activity in S. gallolyticus subsp. gallolyticus UCN34 ΔblpAB and its bWT counterpart containing the reporter plasmid pTCVΩPgllA-gfp with or without addition of 20 nM synthetic GSP. One representative curve of three independent experiments is shown here for each condition. (E) Agar diffusion assay showing gallocin activity in the culture supernatant against S. gallolyticus subsp. macedonicus. Strains tested include S. gallolyticus subsp. gallolyticus UCN34 Δgsp, ΔblpAB and bWT blpAB cultivated with or without addition of 20 nM synthetic GSP.

FIG 2

Mean fluorescence of the reporter strain S. gallolyticus subsp. gallolyticus UCN34 Δgsp pTCVΩPgllA-gfp resuspended in the supernatant of Lactococcus lactis pTCVΩPtetO-blpAB-gsp produced with or without induction with anhydrotetracycline (200 ng/ml) or in THY supplemented with 100 nM synthetic GSP or 200 ng/ml anhydrotetracycline.