Streptococcus suis 2 Transcriptional Regulator TstS Stimulates Cytokine Production and Bacteremia to Promote Streptococcal Toxic Shock-Like Syndrome

Abstract

Two large-scale outbreaks of streptococcal toxic shock-like syndrome (STSLS) have revealed Streptococcus suis 2 to be a severe and evolving human pathogen. We investigated the mechanism by which S. suis 2 causes STSLS. The transcript abundance of the transcriptional regulator gene tstS was found to be upregulated during experimental infection. Compared with the wild-type 05ZY strain, a tstS deletion mutant (ΔtstS) elicited reduced cytokine secretion in macrophages. In a murine infection model, tstS deletion resulted in decreased virulence and bacterial load, and affected cytokine production. Moreover, TstS expression in the P1/7 strain of S. suis led to the induction of STSLS in the infected mice. This is noteworthy because, although it is virulent, the P1/7 strain does not normally induce STSLS. Through a microarray-based comparative transcriptomics analysis, we found that TstS regulates multiple metabolism-related genes and several virulence-related genes associated with immune evasion.

Keywords: STSLS; Streptococcus suis; bacteremia; excessive inflammation; transcriptional regulator.

FIGURE 1

Identification and characterization of the stand-alone transcriptional regulator TstS in the 89K pathogenicity island (PAI). (A) In vivo expression analysis of six stand-alone transcriptional regulators of the 89K PAI in Streptococcus suis. (B) The genetic locus of tstS within the 89K PAI. The arrow shows the direction of transcription and the components were drawn to scale using the Illustrator for Biological Sequences software (Liu et al., 2015).

FIGURE 2

Expression of TstS detected by quantitative polymerase chain reaction (qPCR) in vivo and in human blood. RNA samples from S. suis were obtained from in vitro bacterial cultures, piglet, and mouse blood when hosts presented typical symptoms of S. suis infection, and human blood. The bars represent the standard error of the mean, based on three independent experiments.

FIGURE 3

Cytokines from macrophages were analyzed by qPCR and enzyme-linked immunosorbent assay. Macrophages were stimulated with the strains 05ZY-pSET2, ΔtstS-pSET2, CΔtstS, P1/7-pSET2, or P1/7-tstS at the dose of 5 × 10⁶ CFUs per well for 6 h and then analyzed by qPCR and enzyme-linked immunosorbent assay. (A) The mRNA levels of TNF-α, IL-6, IL-12p35, and IL-12p40 were examined by qPCR. (B) The protein concentrations of cytokines in the culture supernatants were examined by enzyme-linked immunosorbent assay. The bars represent the standard error of the mean, based on three independent experiments. ^∗∗∗P < 0.001; ^∗∗P < 0.01; ^∗P < 0.05.

FIGURE 4

Survival experiments in BALB/c mice. (A) Thirty female BALB/c mice at the age of 5 weeks were randomly assigned to three groups. Mice were intraperitoneally infected with 05ZY-pSET2 (●), ΔtstS-pSET2 (■), or CΔtstS (▲) at a dose of 4 × 10⁸ CFUs per mouse. ^∗∗P < 0.01 for 05ZY-pSET2 versus ΔtstS-pSET2 and ^∗P < 0.05 for ΔtstS-pSET2 versus CΔtstS. (B) Thirty female BALB/c mice at the age of 5 weeks were randomly assigned to three groups equally. The P1/7-pSET2 (▼) and P1/7-tstS (♦) strains were also intraperitoneally administered at a dose of 3 × 10⁸ CFUs per mouse. Animals in the control group were intraperitoneally injected with phosphate-buffered saline (▼). ^∗P < 0.05 for P1/7-pSET2 versus P1/7-tstS CΔtstS. The results shown are representative of three independent experiments.

FIGURE 5

Bacterial counts. (A) Mice were infected with 05ZY-pSET2, ΔtstS-pSET2, or CΔtstS at a dose of 2 × 10⁸ CFUs per mouse. Five randomly selected mice were used for bacterial load analysis. ^∗∗∗∗P < 0.0001; ^∗∗∗P < 0.001; ^∗∗P < 0.01; ^∗P < 0.05. (B) The bacterial loads of P1/7-pSET2 and P1/7-tstS were also measured and compared with those of the 05ZY-pSET2 group. ^∗∗P < 0.01 for P1/7-tstS versus P1/7-pSET2 at 6 h post-inoculation. The bars represent the standard error of the mean, based on three independent experiments.

FIGURE 6

Serum levels of TNF-α, IL-6, and IL-12p70 in BALB/c mice infected with each strain. Five randomly selected mice per group were examined. (A) Mice were infected with 05ZY-pSET2, ΔtstS-pSET2, or CΔtstS at a dose of 2 × 10⁸ CFUs per mouse. ^∗∗∗∗P < 0.0001; ^∗∗∗P < 0.001; ^∗∗P < 0.01; ^∗P < 0.05. (B) Mice were infected with P1/7-pSET2 or P1/7-tstS at a dose of 2 × 10⁸ CFUs per mouse. Data were compared with those from the 05ZY-pSET2 strain. The bars represent the standard errors of the means, based on three independent experiments. ^∗∗∗P < 0.001; ^∗∗P < 0.01; ^∗P < 0.05.

FIGURE 7

TstS influences multiple virulence- and metabolism-related genes. (A) Seven representative genes were selected to determine the correlation between the fold changes (ΔtstS vs. 05ZY) from the microarray and qPCR data. The linear equation was expressed as: Y = 0.97X - 0.1615088 (R² = 0.9064). (B) The column shows the fold changes (ΔtstS vs. 05ZY or P1/7-pSET2 vs. P1/7-tstS) of the seven indicated genes. The primers used for qPCR are listed in Supplementary Table S1. (C) Representative SDS-polyacrylamide gel electrophoresis analysis of purified recombinant TstS. (D) TstS was observed to bind to the SsPep, Fhbp, and Fhb promoters (300 bp) at different concentrations. The SSU05_1809 promoter (300 bp) was used as the negative control.

FIGURE 8

TstS increases the resistance of SS2 to phagocytosis and facilitates the growth of S. suis in blood. Each strain was diluted to 1 × 10⁶ CFUs/ml. Aliquots of the bacterial suspensions (10 μl) were combined with whole blood, complete serum, or inactivated serum (490 μl), and the mixtures were rotated at 37°C. We defined the growth factor as the ratio between the number of CFUs in each sample after incubation divided by the number of CFUs at the first time point (0 h), which was calculated as CFUs_nhours/CFUs_{0 hours}. (A) The growth of each strain in mouse blood. (B) The growth of each strain in complete mouse serum. (C) The growth of each strain in inactivated mouse serum. (D) The survival of each strain in the PMN-mediated bacterial killing assay. The percentage of SS2 that survived was measured as follows: (CFUs_PMN+/CFUs_PMN-) × 100%. (E) The growth of each strain in Dulbecco’s modified Eagle’s medium (DMEM). The bars represent the standard error of the mean, based on three independent experiments. ^∗∗∗P < 0.001; ^∗∗P < 0.01; ^∗P < 0.05.