Investigating the Streptococcus sinensis competence regulon through a combination of transcriptome analysis and phenotypic evaluation

Abstract

Streptococcus sinensis is a recently identified member of the Mitis group of streptococci. This species has been associated with infective endocarditis; however its mechanisms of pathogenesis and virulence are not fully understood. This study aimed to investigate the influence of the competence-stimulating peptide (CSP) and the competence regulon quorum-sensing circuitry (ComABCDE) on subsequent gene transcription and expression, as well as resultant phenotypes. In this study we confirmed the native CSP identity, ascertained when endogenous CSP was produced and completed a transcriptome-wide analysis of all genes following CSP exposure. RNA sequencing analysis revealed the upregulation of genes known to be associated with competence, biofilm formation and virulence. As such, a variety of phenotypic assays were utilized to assess the correlation between increased mRNA expression and potential phenotype response, ultimately gaining insight into the effects of CSP on both gene expression and developed phenotypes. The results indicated that the addition of exogenous CSP aided in competence development and successful transformation, yielding an average transformation efficiency comparable to that of other Mitis group streptococci. Additional studies are needed to further delineate the effects of CSP exposure on biofilm formation and virulence. Overall, this study provides novel information regarding S. sinensis and provides a substantial foundation on which this species and its role in disease pathogenesis can be further investigated.

Keywords: Streptococcus sinensis; competence; competence-stimulating peptide; quorum sensing.

Fig. 1.

Depiction of the ComABCDE QS circuit regulating competence in Mitis group streptococci.

Fig. 2.

(a) Preparative RP-HPLC trace of S. sinensis Forsyth1A spent supernatant following 30 % ammonium sulfate precipitation. (b) Analytical RP-HPLC trace comparing naturally isolated S. sinensis Forsyth1A CSP to synthetic CSP.

Fig. 3.

clustalw alignment of the ComC proteins of several Mitis group streptococci. ComC is presented as a propeptide that is typically cleaved following the double-glycine motif to render the mature CSP signal.

Fig. 4.

RT-qPCR results after 3 min synthetic CSP treatment. Treatment with exogenous CSP for 3 min leads to activation of the comX gene. After a 3 min incubation, stop solution was added to each sample to stop transcription. Data are normalized to expression of gyrB. The RT-qPCR experiment was conducted using two biological replicates for both the CSP and DMSO conditions.

Fig. 5.

Volcano plot of differentially expressed genes comparing treatment with DMSO (negative control) to synthetic CSP. Grey, non-significant genes expressed; red, genes with both a log fold change >2 and cutoff P-value >10⁻⁶ (statistically significant genes).

Fig. 6.

RT-qPCR analysis of original RNA samples used in the RNA sequencing experiment as validation of differential gene expression analysis. After a 3 min incubation, stop solution was added to each sample to stop transcription. Data are normalized to expression of gyrB. The RT-qPCR experiment was conducted using two biological replicates for both the CSP and DMSO conditions.

Fig. 7.

S. sinensis transformation assay. S. sinensis demonstrates dependence on the addition of exogenous synthetic CSP for the successful uptake of added plasmid (pDL278). The synthetic CSP was added at final concentrations of 10 000 nM (a), 1000 nM (b), 100 nM (c) and 10 nM (d). The control includes no exogenous CSP (e). See the Methods section for full experimental details.

Fig. 8.

Effect of exogenous CSP on S. sinensis biofilm formation. The addition of exogenous CSP at varying concentrations does not significantly affect biofilm formation in S. sinensis . The results thereby indicate that the competence regulon QS circuitry in S. sinensis does not seem to have an effect on biofilm formation. See the Supplementary Material for full experimental details.

Fig. 9.

Effect of exogenous CSP on S. sinensis virulence factor production. The addition of exogenous CSP at varying concentrations does not significantly affect lysis of RBCs by S. sinensis . See the Supplementary Material for full experimental details.