The Two-Component Signaling System VraSRss Is Critical for Multidrug Resistance and Full Virulence in Streptococcus suis Serotype 2

Abstract

Streptococcus suis has received increasing attention for its involvement in severe human infections worldwide as well as in multidrug resistance. Two-component signaling systems (TCSSs) play important roles in bacterial adaptation to various environmental stimuli. In this study, we identified a novel TCSS located in S. suis serotype 2 (SS2), designated VraSR_SS, which is involved in bacterial pathogenicity and susceptibility to antimicrobials. Our data demonstrated that the yvqF_SS gene, located upstream of vraSR_SS , shared the same promoter with the TCSS genes, which was directly regulated by VraSR_SS, as shown in electrophoretic mobility shift assays. Notably, YvqF_SS and VraSR_SS constitute a novel multidrug resistance module of SS2 that participates in resistance to certain groups of antimicrobials. Further analyses showed that VraSR_SS inactivation significantly attenuated bacterial virulence in animal models, which, coupled with the significant activation of VraSR_SS expression observed in host blood, strongly suggested that VraSR_SS is an important regulator of SS2 pathogenicity. Indeed, RNA-sequencing analyses identified 106 genes that were differentially expressed between the wild-type and ΔvraSR_SS strains, including genes involved in capsular polysaccharide (CPS) biosynthesis. Subsequent studies confirmed that VraSR_SS indirectly regulated the transcription of CPS gene clusters and, thus, controlled the CPS thickness shown by transmission electron microscopy. Decreased CPS biosynthesis caused by vraSR_SS deletion subsequently increased bacterial adhesion to epithelial cells and attenuated antiphagocytosis against macrophages, which partially clarified the pathogenic mechanism mediated by VraSR_SS Taken together, our data suggest that the novel TCSS, VraSR_SS, plays critical roles for multidrug resistance and full virulence in SS2.

Keywords: Streptococcus suis; capsular biosynthesis; multidrug resistance; two-component signaling system; virulence.

FIG 1

Identification of a novel TCSS in SS2 strain ZY05719. (A) Homology analyses of the ZY05719_02075, ZY05719_02080, and ZY05719_02085 proteins by the protein BLAST algorithm. The identities of the amino acid sequences encoded by each gene are shown between SS2 strain ZY05719 and S. aureus strain Mu50. (B) Predicted three-dimensional structure and conserved domains of the ZY05719_02080 protein, as determined using the SWISS-MODEL online server. The HAMP domain that potentially mediates transmembrane signal transduction, the DHp domain containing a conserved histidine residue, and the CA domain were identified by sequence alignment and are labeled accordingly. (C) Three-dimensional structure and conserved domains of the ZY05719_02085 protein, predicted using the SWISS-MODEL online server. A REC domain catalyzing phosphoryl and an HTH domain used for DNA binding were identified by sequence alignment and are labeled accordingly.

FIG 2

VraSR_SS directly regulated the expression of YvqF_SS and itself in SS2. (A) The yvqF_SS, vraS_SS, and vraR_SS genes formed an operon, as determined by RT-PCR. An RNA sample that was not reverse transcribed served as a negative control. (B) Expression levels of yvqF_SS in the WT, ΔvraSR_SS, and complemented strains were measured by qRT-PCR. qRT-PCR expression values are shown as the means plus standard deviations (error bars) from at least three independent experiments. The unpaired two-tailed Student's t test was used for statistical analysis (ns, P > 0.05; ***, P < 0.001). (C) An EMSA showing VraR_SS binding to the promoter region of the yvqF_SS operon. The purified VraR_SS fusion protein was added to each reaction mixture at different concentrations. DNA probes containing the yvqF_SS operon promoter region were used at 80 ng per reaction mixture, and fragments amplified from 16S rRNA served as a negative control.

FIG 3

VraSR_SS contributed to the full virulence of SS2. (A) Levels of vraSR_SS gene expression during THB culture (in vitro) and animal infection (mouse blood, in vivo) were analyzed by qRT-PCR. The unpaired two-tailed Student's t test was used for statistical analysis (***, P < 0.001). (B) Deletion of vraSR_SS resulted in significant attenuation of mortality in SS2 infection. Randomized groups of 10 SPF BALB/c mice were challenged with different bacterial strains at a dose of 5 × 10⁸ CFU/mouse. Another 10 SPF BALB/c mice injected with PBS served as negative controls. The survival rates were monitored for 7 days after challenge. Significant differences in survival between different groups were analyzed by log-rank (Mantel-Cox) test (P < 0.05).

FIG 4

Identification of VraSR_SS-regulated genes via transcriptional analysis. (A) Comparison of gene regulation by RNA-Seq analysis or qRT-PCR. qRT-PCR was used to validate the expression level changes of 10 selected genes, including 4 upregulated genes and 6 downregulated genes, identified by RNA-Seq analysis. Each sample was run in triplicate, and the reference gene parC was detected as a control. Relative fold changes in expression were calculated using the 2^−ΔΔCT method. (B) The VraSR_SS-regulated genes identified by transcriptional analysis were involved in CPS biosynthesis. The expression levels of CPS biosynthesis-related genes were significantly lower in the ΔvraSR_SS strain.

FIG 5

VraSR_SS indirectly regulated expression of the CPS biosynthesis gene cluster in SS2. (A) Schematic diagram of the genetic organization of the CPS synthesis and the sialic acid synthase clusters. The arrows indicate the direction of transcription. (B) Expression levels of cps2C, cps2F, and neuB in ZY05719, ΔvraSR_SS, and CΔvraSR_SS strains were measured by qRT-PCR. The unpaired two-tailed Student's t test was used for statistical analysis (ns, P > 0.05; ***, P < 0.001). (C) Transmission electron micrographs of the ZY05719, ΔvraSR_SS, and CΔvraSR_SS strains. The scale bars indicate the magnification size. (D) Photomicrographs of the capsules of the ZY05719, ΔvraSR_SS, and CΔvraSR_SS strains stained with crystal violet (1,000×). (E) VraR_SS indirectly regulated the CPS biosynthesis gene cluster. Either the cps or neu promoter region could be shifted by the recombinant VraR_SS protein in EMSAs. DNA probes containing the yvqF_SS operon promoter region were used as a positive control, and fragments amplified from 16S rRNA served as a negative control.

FIG 6

VraSR_SS is involved in SS2 adhesion to bEnd.3 cells and resisting phagocytosis by RAW 264.7 cells. (A) Effect of the vraSR_SS mutation on the ability of SS2 to adhere to bEnd.3 cells. The adhesion rate of the ZY05719 strain was significantly lower than that of the ΔvraSR_SS and Δcps strains. The vraSR_SS-complemented strain showed adhesion restored to the level of the WT strain. (B) Effect of the vraSR_SS deletion on the ability of SS2 to resist phagocytosis by RAW 264.7 cells. The antiphagocytosis rate of the ZY05719 strain was significantly higher than that of the ΔvraSR_SS and Δcps strains. The vraSR_SS-complemented strain showed an ability restored to the level of the WT strain. The data are shown as the means and standard deviations of the results from three independent experiments performed in triplicate. Two-tailed unpaired Student's t tests were used for statistical analysis (***, P < 0.001).