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

Abstract

Streptococcus mutans, a causative agent of dental caries, relies on multiple quorum-sensing (QS) pathways that coordinate the expression of factors needed for colonization in the oral cavity. S. mutans uses small peptides as QS signaling molecules that typically are secreted into the outside milieu. Competence-stimulating peptide (CSP) is one such QS signaling molecule that functions through the ComDE two-component signal transduction pathway. CSP is secreted through NlmTE, a dedicated ABC transporter that cleaves off the N-terminal leader peptide to generate a mature peptide that is 21 residues long (CSP-21). We recently identified a surface-localized protease, SepM, which further cleaves the CSP-21 peptide at the C-terminal end and removes the last 3 residues to generate CSP-18. CSP-18 is the active QS molecule that interacts with the ComD sensor kinase to activate the QS pathway. In this study, we show that SepM specifically cleaves CSP-21 between the Ala18 and Leu19 residues. We also show that SepM recognizes only Ala at position 18 and Leu at position 19, although some CSP-18 variants with a substitution at position 18 can function equally as well as the QS peptide. Furthermore, we demonstrate that SepM homologs from other streptococci are capable of processing CSP-21 to generate functional CSP-18. IMPORTANCESepM is a membrane-associated streptococcal protease that processes competence-stimulating peptide (CSP) to generate an active quorum-sensing molecule in S. mutans. SepM belongs to the S16 family of serine proteases, and in this study, we found that SepM behaves as an endopeptidase. SepM displays strict substrate specificity and cleaves the peptide bond between the Ala and Leu residues. This is the first report of an endopeptidase that specifically cleaves these two residues. S treptococcus mutans is considered to be one of the primary etiological agents of dental caries. S. mutans forms robust biofilms on tooth surfaces and heart valves, a prerequisite for disease formation. Biofilm formation on the tooth surface requires a highly coordinated signaling pathway known as quorum sensing (QS). QS is a primary bacterial communication system that often uses secreted peptide pheromones to regulate the expression of various genes when the bacterial cell density reaches a certain threshold concentration (1). In addition to biofilm formation, numerous cellular processes, such as virulence factor expression, extracellular enzyme production, antibiotic production, and genetic exchanges, are coordinated by QS (2, 3). S. mutans employs a well-conserved QS system called ComDEC, which is required for the regulation of biofilm formation, stress responses, the expression of bacteriocin-encoding genes, and the development of genetic competence (4). S. mutans and other Gram-positive bacteria generally use peptides as QS molecules (5). These peptides typically are translated as prepeptides that undergo processing during export to the extracellular environment. In S. mutans, the peptide pheromone competence-stimulat...

Streptococcus endopeptidases promote HPV infection in vitro

“…The SGO_0652 locus encodes a 347‐amino acid with a calculated molecular weight of 38.1 kDa. Without a signal peptide, this locus is homologous to the conserved sepM gene in S. mutans , which encodes a membrane‐associated peptidase required for processing competence stimulating peptide (CSP) for competence development and quorum sensing (Biswas, Cao, Kim, & Biswas, ; Hossain & Biswas, ). With one transmembrane domain, S. gordonii sepM is also predicted as a membrane‐associated peptidase.…”

“…SepM, a cell membrane‐associated peptidase, is required for the CSP maturation in S. mutans (Biswas et al., ; Hossain & Biswas, ). SepM proteins from S. mutans, S. pyogenes and S. agalactiae all activate the S. mutans CSP by cleaving CSP‐21 between the Ala18 and Leu19 residues, but SepM from S. gordonii does not (Biswas et al., ).…”

“…Because inactivation of pepO substantially reduced competence in S. gordonii , the intended function of PepO might be associated with competence development. Although S. gordonii pepO expression is not induced by CSP (Vickerman et al., ), at least two peptidases in Streptococcus , ComA (Ishii et al., ) and SepM (Biswas et al., ), have been reported to promote competence by processing CSP to its mature form. The mechanism by which S. gordonii PepO facilitates genetic transformation is unknown, but it appears to play this role intracellularly, because adding purified PepO to competent pepO mutant culture did not restore its transformation to the level of the wild type.…”

“…These mutants showed further reduced furin-like activity compared with the wild-type and single-gene knockout mutants (Figure 1c), suggesting that these enzymes have redundant activities. Among the three proteins, none contain a classical signal peptide for secretion, and since the secA2 mutation ( locus is homologous to the conserved sepM gene in S. mutans, which encodes a membrane-associated peptidase required for processing competence stimulating peptide (CSP) for competence development and quorum sensing (Biswas, Cao, Kim, & Biswas, 2015;Hossain & Biswas, 2012). With one transmembrane domain, S. gordonii sepM is also predicted as a membrane-associated peptidase.…”

“…SepM, a cell membrane-associated peptidase, is required for the CSP maturation in S. mutans (Biswas et al, 2015;Hossain & Biswas, 2012 Figure 1a).…”