An Extracelluar Protease, SepM, Generates Functional Competence-Stimulating Peptide in Streptococcus mutans UA159

Abstract

Cell-cell communication in Gram-positive bacteria often depends on the production of extracellular peptides. The cariogenic bacterium Streptococcus mutans employs so-called competence-stimulating peptide (CSP) to stimulate mutacin (bacteriocin) production and competence development through the activation of the ComDE two-component pathway. In S. mutans, CSP is secreted as a 21-residue peptide; however, mass spectrometric analysis of culture supernatant indicates the presence of an 18-residue proteolytically cleaved species. In this study, using a transposon mutagenesis screening, we identified a cell surface protease that is involved in the processing of 21-residue CSP to generate the 18-residue CSP. We named this protease SepM for streptococcal extracellular protease required for mutacin production. We showed that the truncated 18-residue peptide is the biologically active form and that the specific postexport cleavage is a prerequisite to activate the ComDE two-component signal transduction pathway. We also showed that the CSP and the mutacins are exported outside the cell by the same ABC transporter, NlmTE. Our study further confirmed that the ComDE two-component system is absolutely necessary for mutacin production in S. mutans. Quorum sensing is a primary means of bacterial communication that often uses secreted peptide pheromones to regulate expression of various genes when the bacterial cell density reaches a certain threshold concentration. Numerous cellular processes, such as virulence factor expression, extracellular enzyme production, antibiotic production, biofilm formation, and genetic exchange, are regulated by quorum sensing (25,38,48,54,55). In streptococci, competence-stimulating peptide (CSP)-mediated quorum sensing is a prerequisite for development of competence that leads to cellular DNA uptake from the surroundings for genetic diversity (41). The Gram-positive pathogen Streptococcus mutans, a primary causative agent of dental caries, employs a wellconserved quorum-sensing system called ComCDE that coordinates the expression of bacteriocins encoding genes and stimulates development of genetic competence (22,23,30,40,51,57). The ComCDE-regulated quorum-sensing system has also been implicated in regulation of biofilm formation (30) and stress responses (28) in addition to other cellular processes.Gram-positive bacteria use ribosomally synthesized peptides as quorum-signaling molecules (31). These peptides are typically translated as prepeptides that undergo processing during export to the extracellular environment (48). In S. mutans UA159, the peptide pheromone CSP is encoded by comC as a prepeptide with a leader sequence containing a conserved double glycine (GG) motif. During secretion through a dedicated ABC transporter complex, the N-terminal leader peptide is cleaved off by the proteolytic activity of the transporter to generate a mature peptide that is 21 residues long (CSP-21) (15,30,43). When the extracellular CSP concentration reaches a certain threshold, ComD, a membrane-associated hi...

SmbFT, a Putative ABC Transporter Complex, Confers Protection against the Lantibiotic Smb in Streptococci

“…Plasmid pIB184-Km was used as vector for cloning (47). This plasmid contains P23 promoter from lactococcal phage (pOri23).…”

“…Inactivation of smbAB and smbT genes in GS-5. A previously described fusion PCR method was used for the construction of ⌬smbAB and ⌬smbT mutant strains (47). In short, 500-bp upstream (up) and downstream (dn) flanking regions of the respective genes were separately PCR amplified using GS-5 genomic DNA as a template (see Table 1 for the primers).…”

SepM, a Streptococcal Protease Involved in Quorum Sensing, Displays Strict Substrate Specificity

“…Early reports indicated that CSP-18 is more potent and works at a much lower concentration (10-fold) than CSP-21 (13). We recently identified a membrane-associated protease called SepM, which is responsible for the processing of CSP-21 to generate the active QS peptide CSP-18 (8). Surprisingly, genome analysis suggests that S. mutans clinical isolates secrete various CSP subtypes, although the variation among them is not as diverse as that of the pneumococcus CSP (14)(15)(16).…”

“…SepM contains at least three domains: a transmembrane domain spanning from residues 10 to 26, a eukaryotic-domain-like PDZ domain spanning residues 131 to 195, and a Lon-like proteolytic domain at the C-terminal end (8). As with the Lon protease, this S16 type protease SepM contains a Ser-Lys dyad (S235 and K280) in its active site, in which serine is the nucleophile.…”

“…In S. mutans, the peptide pheromone competence-stimulating peptide (CSP) is encoded by the comC gene as a prepeptide with a leader sequence containing a conserved double-glycine (GG) motif (4,6). During secretion through NlmTE, a dedicated ABC transporter complex, the N-terminal leader peptide, is cleaved off by the proteolytic activity of the transporter to generate a mature peptide that is 21 residues long (CSP-21) (7,8). When the extracellular CSP concentration reaches a certain threshold, ComD, a membrane-associated histidine kinase, senses the signal.…”