Transcriptional Profiling of the Oral Pathogen Streptococcus mutans in Response to Competence Signaling Peptide XIP

Abstract

In the cariogenic Streptococcus mutans, competence development is regulated by the ComRS signaling system comprised of the ComR regulator and the ComS prepeptide to the competence signaling peptide XIP (ComX-inducing peptide). Aside from competence development, XIP signaling has been demonstrated to regulate cell lysis, and recently, the expression of bacteriocins, small antimicrobial peptides used by bacteria to inhibit closely related species. Our study further explores the effect of XIP signaling on the S. mutans transcriptome. RNA sequencing revealed that XIP induction resulted in a global change in gene expression that was consistent with a stress response. An increase in several membrane-bound regulators, including HdrRM and BrsRM, involved in bacteriocin production, and the VicRKX system, involved in acid tolerance and biofilm formation, was observed. Furthermore, global changes in gene expression corresponded to changes observed during the stringent response to amino acid starvation. Effects were also observed on genes involved in sugar transport and carbon catabolite repression and included the levQRST and levDEFG operons. Finally, our work identified a novel heat shock-responsive intergenic region, encoding a small RNA, with a potential role in competence shutoff. IMPORTANCE Genetic competence provides bacteria with an opportunity to increase genetic diversity or acquire novel traits conferring a survival advantage. In the cariogenic pathogen Streptococcus mutans, DNA transformation is regulated by the competence stimulating peptide XIP (ComX-inducing peptide). The present study utilizes high-throughput RNA sequencing (RNAseq) to provide a greater understanding of how global gene expression patterns change in response to XIP. Overall, our work demonstrates that in S. mutans, XIP signaling induces a response that resembles the stringent response to amino acid starvation. We further identify a novel heat shock-responsive intergenic region with a potential role in competence shutoff. Together, our results provide further evidence that multiple stress response mechanisms are linked through the genetic competence signaling pathway in S. mutans.

Keywords: ComRS; Streptococcus mutans; XIP; cell signaling; genetic competence; signaling peptides; transcriptome.

FIG 1

Functional characterization of the changes (>2-fold) in the S. mutans UA159 transcriptome in response to 1 µM XIP. Genes up- or downregulated by >2-fold were grouped on the basis of their annotated function, and their fold change values were graphed.

FIG 2

Effects of increasing amounts of XIP on genes involved in bacteriocin regulation (a) and sugar uptake (b). RT-PCR analysis of comC, comD, and comE (a) or levR, levRD, and manL (b) gene expression was performed in strain UA159 grown in CDM to an OD₆₀₀ of 0.4 in the presence of increasing amounts of XIP. Values that are significantly different from the value for the DMSO control are indicated by asterisks as follows: *, P < 0.05; **, P < 0.005.

FIG 3

Functional characterization of the changes (>2-fold) in the S. mutans ΔSMcomS transcriptome in response to 1 µM XIP. Genes up- or downregulated by >2-fold were grouped on the basis of their annotated function, and their fold change values were graphed.

FIG 4

Expression of selected genes in the ΔSMcomS strain in response to increasing concentrations of XIP. RT-PCR analysis of gene expression was performed in ΔSMcomS grown in CDM to an OD₆₀₀ of 0.4 in the presence of increasing amounts of XIP. *, P < 0.05; **, P < 0.005.

FIG 5

Expression of selected intergenic regions in response to increasing concentrations of XIP. qRT-PCR analysis of gene expression was performed with strain UA159 grown in CDM to an OD₆₀₀ of 0.4 in the presence of increasing amounts of XIP. *, P < 0.05; ***, P < 0.001.

FIG 6

Effect of ΔSMU.82-83 on transformation frequency. Overnight cultures of S. mutans UA159 and ΔSMU.82-83 strains were diluted 1:20 in fresh THYE and incubated in the presence or absence of pDL277 (Spec^r). The resulting percent transformation frequencies are shown. Values are means plus standard errors (error bars) from three independent experiments.

FIG 7

Overlap of the XIP transcriptomes in S. mutans UA159 and ΔSMcomS strains. A Venn diagram summarizing the overlap between upregulated (a) or downregulated (b) genes in UA159 and ΔSMcomS strains.