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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 ?clpP 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 ?clpX strain, 113 (47?%) of which also appeared in the ?clpP microarrays. Several genes associated with cell growth were downregulated in both mutants, consistent with the slow-growth phenotype of the ?clp 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 ?clpP and ?clpX 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 ?clp 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 ?clp strains. Collectively, our results reveal the broad scope of ClpXP regulation in S. mutans homeostasis and identify several virulence-related traits that are influenced by ClpXP proteolysis.

SUBMITTER: Kajfasz JK

PROVIDER: S-EPMC3353396 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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

Kajfasz Jessica K JK Abranches Jacqueline J Lemos José A JA

Microbiology (Reading, England) 20110804 Pt 10

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 ΔclpP strain, 288 genes showed significant changes in relative transcript amou ...[more]

PMID: 21816882

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Project description:Streptococcus mutans, a dental pathogen, has a remarkable ability to cope with environmental stresses. Under stress conditions, cytoplasmic proteases play a major role in controlling the stability of regulatory proteins and preventing accumulation of damaged and misfolded proteins. ClpXP, a well-conserved cytoplasmic proteolytic system, is crucial in maintaining cellular homeostasis in bacteria. ClpX is primarily responsible for recognition of substrates and subsequent translocation of unfolded substrates into the ClpP proteolytic compartment for degradation. In Escherichia coli, ClpX recognizes distinct motifs present at the C-terminal end of target proteins. However, recognition sequences for ClpXP in other bacteria, including S. mutans, are not known. In this study, using two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) analysis, we have identified several putative substrates for S. mutans ClpXP. SsbA, which encodes a small DNA binding protein, is one such substrate that is degraded by ClpXP. By sequential deletions, we found that the last 3 C-terminal amino acids, LPF, are sufficient for ClpXP-mediated degradation. Addition of LPF at the C-terminal end of green fluorescent protein (GFP) rendered the protein completely degradable by ClpXP. Alterations of this tripeptide motif impeded ClpXP-mediated degradation. However, recognition of LPF by ClpXP is highly specific to some S. mutans strains (UA159, UA130, and N3209) since not all S. mutans strains recognize the motif. We speculate that an adaptor protein is involved in either substrate recognition or substrate degradation by ClpXP. Nevertheless, this is the first report of a unique recognition sequence for ClpXP in streptococci. IMPORTANCE Regulated proteolysis in bacteria is an important biological process that maintains protein homeostasis. ClpXP, an intracellular proteolytic complex, is the primary protease that is responsible for protein turnover. While the substrates for ClpXP were identified in Escherichia coli, the substrates for vast majority of bacteria are currently unknown. In this study, we identified a unique substrate for ClpXP-mediated degradation in Streptococcus mutans, a dental pathogen. We also found that a small motif composed of 3 amino acids is sufficient for ClpXP-mediated degradation. Identification of this motif will clearly help us to understand the pathogenesis of this organism and other related pathogens.

Project description:Streptococcus mutans is a key cariogenic bacterium responsible for the initiation of tooth decay. Biofilm formation is a crucial virulence property. We discovered a putative glycosyltransferase, SMU_833, in S. mutans capable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion of smu_833 decreases glucan and increases eDNA but maintains the overall biofilm biomass. The decrease in glucan is caused by a reduction in GtfB and GtfC, two key enzymes responsible for the synthesis of glucan. The increase in eDNA was accompanied by an elevated production of membrane vesicles, suggesting that SMU_833 modulates the release of eDNA via the membrane vesicles, thereby altering biofilm matrix constituents. Furthermore, glucan and eDNA were colocalized. The complete deletion of gtfBC from the smu_833 mutant significantly reduced the biofilm biomass despite the elevated eDNA, suggesting the requirement of minimal glucans as a binding substrate for eDNA within the biofilm. Despite no changes in overall biofilm biomass, the mutant biofilm was altered in biofilm architecture and was less acidic in vitro Concurrently, the mutant was less virulent in an in vivo rat model of dental caries, demonstrating that SMU_833 is a new virulence factor. Taken together, we conclude that SMU_833 is required for optimal biofilm development and virulence of S. mutans by modulating extracellular matrix components. Our study of SMU_833-modulated biofilm matrix dynamics uncovered a new target that can be used to develop potential therapeutics that prevent and treat dental caries.IMPORTANCE Tooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacterium Streptococcus mutans The bacteria utilize dietary sugars to build and strengthen biofilms, trapping acids onto the tooth's surface and causing demineralization and decay of teeth. As knowledge of our body's microbiomes increases, the need for developing therapeutics targeted to disease-causing bacteria has arisen. The significance of our research is in studying and identifying a novel therapeutic target, a dynamic biofilm matrix that is mediated by a new virulence factor and membrane vesicles. The study increases our understanding of S. mutans virulence and also offers a new opportunity to develop effective therapeutics targeting S. mutans In addition, the mechanisms of membrane vesicle-mediated biofilm matrix dynamics are also applicable to other biofilm-driven infectious diseases.

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

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

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