Transcriptome analysis reveals that ClpXP proteolysis controls key virulence properties of Streptococcus mutans

Abstract

The ClpXP proteolytic complex is critical for maintaining cellular homeostasis, as well as expression of virulence properties. However, with the exception of the Spx global regulator, the molecular mechanisms by which the ClpXP complex exerts its influence in Streptococcus mutans are not well understood. Here, microarray analysis was used to provide novel insights into the scope of ClpXP proteolysis in S. mutans. In a DclpP strain, 288 genes showed significant changes in relative transcript amounts (P¡0.001, twofold cut-off) as compared with the parent. Similarly, 242 genes were differentially expressed by a DclpX strain, 113 (47 %) of which also appeared in the DclpP microarrays. Several genes associated with cell growth were downregulated in both mutants, consistent with the slow-growth phenotype of the Dclp strains. Among the upregulated genes were those encoding enzymes required for the biosynthesis of intracellular polysaccharides (glg genes) and malolactic fermentation (mle genes). Enhanced expression of glg and mle genes in DclpP and DclpX strains correlated with increased storage of intracellular polysaccharide and enhanced malolactic fermentation activity, respectively. Expression of several genes known or predicted to be involved in competence and mutacin production was downregulated in the Dclp strains. Follow-up transformation efficiency and deferred antagonism assays validated the microarray data by showing that competence and mutacin production were dramatically impaired in the Dclp strains. Collectively, our results reveal the broad scope of ClpXP regulation in S. mutans homeostasis and identify several virulencerelated traits that are influenced by ClpXP proteolysis. INTRODUCTIONStreptococcus mutans is a member of the oral microbiome known for its close association with dental caries and, occasionally, infective endocarditis. The niche in which S. mutans thrives is the biofilm that forms on the enamel surface of teeth (Loesche, 1986). The dental biofilm environment is constantly and unpredictably changing due to the eating habits of the human host, resulting in large fluctuations in nutrient source and availability, pH, and oxygen tension, among other stresses . The remarkable ability of S. mutans to tolerate and thrive during stressful conditions, particularly low pH, is closely linked to its virulence in the oral cavity.The Clp proteolytic complex is critical in maintaining cellular homeostasis, particularly for organisms that must continually endure environmental fluctuations (Frees et al., 2004;Gottesman, 2003;Jenal & Hengge-Aronis, 2003; Kajfasz et al., 2009). In S. mutans, Clp proteases are the result of the association of the ClpP peptidase with one of several Clp ATPases (ClpC, ClpE or ClpX), forming barrelshaped complexes that will target proteins for degradation (Kajfasz et al., 2009;Lemos & Burne, 2002). Although S. mutans also encodes ClpB and ClpL ATPases, these proteins do not contain the recognition tripeptide that permits interaction with ClpP, and are believed to function mainly...

Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology

“…In fact, this is the case in S. thermophilus where CRISPR-Cas systems were observed to function independently (86). Furthermore, several transcriptome studies revealed that deletion of virulence or global regulatory genes of S. mutans (including genes involved in stress response) differently affected transcription of cas genes within CRISPR-Cas systems, suggesting different roles for cas genes within the cell (50,54,87). As already proven in other systems (88-91), it is possible that different regulatory systems, in addition to VicR/K, interact with S. mutans CRISPR-Cas systems to mediate gene expression in response to cues such as oxidative stress and cell membrane changes or alterations in the internal pH of the cell.…”

“…Subsequently, it was shown that M102 adheres to phage-sensitive and phage-resistant S. mutans serotype c strains, indicating that factors besides phage adsorption determine resistance of S. mutans serotype c strains to infection by M102 phage (49). Despite these studies that explored the role of CRISPR-Cas systems in S. mutans in conferring phage immunity, recent transcriptome studies suggest other functions that CRISPR-Cas systems might have in S. mutans are poorly understood (50)(51)(52)(53)(54)(55).…”

Stress Physiology of Lactic Acid Bacteria

“…Although ClpL is required for growth at 43°C and for penicillin tolerance (478,483), an S. pneumoniae ⌬clpL mutant showed a level of virulence similar to that of the parental strain in a murine intraperitoneal infection model (478). Systematic deletion of clpP and the five Clp ATPase genes (clpB, clpC, clpE, clpL, and clpX) in the dental pathogen S. mutans revealed that the ClpXP proteolytic system modulates the expression of several virulence-related traits (304,484) and that the infectivity of strains lacking clpP or clpX is slightly reduced in a rat model of oral colonization (304). Inactivation of the remaining Clp ATPases did not affect tooth colonization, but the role of the S. mutans Clp proteins in caries development or in infective endocarditis remains to be evaluated further.…”

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

“…In Streptococcus mutans, the causative agent of tooth decay, mutations in virulence or global regulatory genes (including genes involved in stress responses) strongly affect gene transcription (81)(82)(83)(84)(85), including that of the type II cas genes ( Fig. 1 and 4).…”