Regulation and Physiological Significance of ClpC and ClpP in Streptococcus mutans

Abstract

Tolerance of environmental stress, especially low pH, by Streptococcus mutans is central to the virulence of this organism. The Clp ATPases are implicated in the tolerance of, and regulation of the response to, stresses by virtue of their protein reactivation and remodeling activities and their capacity to target misfolded proteins for degradation by the ClpP peptidase. The purpose of this study was to dissect the role of selected clp genes in the stress responses of S. mutans, with a particular focus on acid tolerance and adaptation. Homologues of the clpB, clpC, clpE, clpL, clpX, and clpP genes were identified in the S. mutans genome. The expression of clpC and clpP, which were chosen as the focus of this study, was induced at low pH and at growth above 40°C. Inactivation of ctsR, the first of two genes in the clpC operon, demonstrated that CtsR acts as a repressor of clp and groES-EL gene expression. Strains lacking ClpP, but not strains lacking ClpC, were impaired in their ability to grow under stress-inducing conditions, formed long chains, aggregated in culture, had reduced genetic transformation efficiencies, and had a reduced capacity to form biofilms. Comparison of two-dimensional protein gels from wild-type cells and the ctsR and clpP mutants revealed many changes in the protein expression patterns. In particular, in the clpP mutant, there was an increased production of GroESL and DnaK, suggesting that cells were stressed, probably due to the accumulation of denatured proteins.

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

“…Standard DNA manipulation techniques were used as previously described (Lemos & Burne, 2002;Sambrook et al, 1989). The primers used to isolate the mutants are listed in Supplementary Table S1.…”

“…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 as molecular chaperones (Frees et al, 2004; Kajfasz et al, 2009 Although ClpP proteolysis has been consistently implicated in virulence (Frees et al, 2003;Gaillot et al, 2000;Ibrahim et al, 2005), has shown promising results as a vaccine candidate (Kwon et al, 2004) and is the target of two new classes of antibiotics (Böttcher & Sieber, 2008; Brötz-Oesterhelt et al, 2005), a complete picture of the biological role of Clp-dependent proteolysis in bacterial pathogenesis has yet to emerge.…”

“…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 as molecular chaperones (Frees et al, 2004;Kajfasz et al, 2009).…”

Stress Physiology of Lactic Acid Bacteria

“…Phenotypic studies with mutant strains lacking functional ClpP, ClpL, ClpE, or ClpB revealed the central role of these proteins under stress conditions and, for pathogenic LAB species, in virulence (303,(305)(306)(307)(308). In contrast to the situation in B. subtilis, LAB clpC mutants usually do not exhibit stress-sensitive phenotypes (306,309). Phenotypic characterization of an S. thermophilus clpC mutant revealed that ClpC plays a role in the development of genetic competence in this organism (310).…”

Molecular Genetic Analysis of the Virulence of Oral Bacterial Pathogens: An Historical Perspective

“…Indirectly, this can be considered as a genetic approach, since the identification of the proteins on the gels is made based upon a comparison with the protein sequences deduced from the genome sequences of an organism. This approach has been utilized in several recent investigations regarding the effects of various environmental factors on S. mutans (Svensater et al, 2000;Lemos and Burne, 2002).…”