An Auto-Regulating Type II Toxin-Antitoxin System Modulates Drug Resistance and Virulence in Streptococcus suis

Abstract

Toxin-antitoxin (TA) systems are ubiquitous genetic elements that play an essential role in multidrug tolerance and virulence of bacteria. So far, little is known about the TA systems in Streptococcus suis. In this study, the Xress-MNTss TA system, composed of the MNTss toxin in the periplasmic space and its interacting Xress antitoxin, was identified in S. suis. β-galactosidase activity and electrophoretic mobility shift assay (EMSA) revealed that Xress and the Xress-MNTss complex could bind directly to the Xress-MNTss promoter as well as downregulate streptomycin adenylyltransferase ZY05719_RS04610. Interestingly, the Xress deletion mutant was less pathogenic in vivo following a challenge in mice. Transmission electron microscopy and adhesion assays pointed to a significantly thinner capsule but greater biofilm-formation capacity in ΔXress than in the wild-type strain. These results indicate that Xress-MNTss, a new type II TA system, plays an important role in antibiotic resistance and pathogenicity in S. suis.

Keywords: Streptococcus suis; antitoxin; drug resistance; toxin; virulence.

FIGURE 1

Effect of toxin and antitoxin induction on growth of E. coli Top10 cells. Toxins were linked to pBADHisA and pBADHisA-pelB plasmids and transformed into E. coli Top10 cells. Cytoplasmic toxicity in cells expressing the toxin on pBADHisA (A) with 0.2% L-arabinose. Periplasmic toxicity in cells expressing the toxin on pBADHisA-pelB (B) with L-arabinose. (C) Viability of strains expressing T2/T3, MNTss, T6, and RHSse. To quantitate CFU per milliliter, cells were diluted and plated on LB agar plus 100 ug of ampicillin/ml at the times indicated. Growth of E. coli Top10 cells containing the pBADHisA-pelB-Xress-MNTss plasmid with 0.2% L-arabinose (D) to determine the neutralization effect of the antitoxin. Culture growth was monitored by measuring OD₆₀₀ every hour. Growth curves represent at least three independent experiments.

FIGURE 2

Evaluation of relative expression and promoter activity in vivo. (A) Predicted Xress-MNTss promoter. Gray areas comprise the -35 box and -10 box regions, blue arrows represent the predicted palindrome sequences. P indicates the transcriptional start site (TSS). (B) Expression levels of MNTss and Xress in ZY05719, ΔXress-MNTss, ΔMNTss, and ΔXress strains as measured by qRT-PCR. The relative expression levels represented the mean ± SD of three biological repeats. (C) The pTCV-lac reporter plasmid (pTCV-lac-300), containing the Xress-MNTss promoter sequence, was transferred to ΔXress-MNTss,CΔXress-MNTss and ZY05719 to determine β-galactosidase activity. (D) (i) Toxin point mutant D-MNTss was constructed by mutating the start codon ATG of MNTss to CTG; (ii) antitoxin point mutant D-Xress was constructed by mutating the start codon ATG of Xress to CTG. (E) Promoter assay using pTCV-lac-300 integrated in the D-MNTss and D-Xress host. The data are shown as the means and standard deviations of the results from three independent experiments performed in triplicate. Unpaired two-tailed Student’s t-test: ns, P > 0.05; ^∗∗∗P < 0.001; ^****P < 0.0001.

FIGURE 3

EMSA analysis in vitro. (A) Purification of Xress-His protein from pCold^TM II-Xress-containing BL21(DE3) E. coli. (B) Purification of Xress-MNTss-His protein from pCold^TM II-Xress-MNTss containing BL21(DE3) E. coli. (C) EMSA result showing binding of the antitoxin Xress to the Xress-MNTss promoter. The purified Xress protein was added to each reaction mixture at different concentrations. DNA probes containing the Xress-MNTss operon promoter region were used at 80 ng per reaction mixture (lanes 1–5). And fragments amplified from 16S rRNA served as a negative control (lanes 6–7). (D) TA Xress-MNTss complex bound to the Xress-MNTss promoter. The Xress-MNTss promoter region could be shifted by the TA complex in EMSA. DNA probes containing the Xress-MNTss operon promoter region were used at 80 ng per reaction mixture (lanes 1–4). And fragments amplified from 16S rRNA served as a negative control (lanes 5–6).

FIGURE 4

Xress-MNTss regulates the zy05719_RS05610 gene. (A) Putative genomic island genes annotation. (B) ZY05719_RS04595, zy05719_RS04600, zy05719_RS04605, and zy05719_RS04610 formed an operon, as determined by RT-PCR. An RNA sample without reverse transcription served as a negative control. (C) Expression of zy05719_RS04610 in ZY05719, ΔXress-MNTss, ΔMNTss, and ΔXress strains as measured by qRT-PCR. Values are shown as the means plus standard deviations (error bars) from at least three independent experiments. Unpaired two-tailed Student’s t-test: P < 0.0001.

FIGURE 5

Xress-MNTss contributes to S. suis virulence and affects biofilm formation. (A) Survival curves of 5-week-old BALB/c mice infected with wild-type or mutant strains at 5 × 10⁸ CFU/mouse. The control group received only PBS. Ten mice from each group were monitored over a 7-day period. Log-rank (Mantel-Cox) test to determine differences in survival between groups: ^∗∗P < 0.01. (B) Infected mice were euthanized 6 h after infection to determine the bacteria burden in the blood, brain, liver, and spleen. Biofilm formation in panel (C,D) 200 μl culture medium was inoculated in each well of 96-well plate. Unpaired two-tailed Student’s t-test: ^****P < 0.0001.

FIGURE 6

Capsule synthesis is reduced in the ΔXress mutant. (A) Adhesion of ΔXress to Hep-2 epithelial cells. The adhesion rate of the ZY05719 strain was significantly lower than that of the ΔXress strain. Values represent the average of three independent replicates. Two-tailed unpaired Student’s t-test: ^∗P < 0.05. (B) Dot-blot membrane showing capsule productions in ZY05719, ΔCPS (control), and ΔXress. Dilutions of cells were made in PBS and spotted onto nitrocellulose membranes; the cells were then fixed and the membranes were probed with the absorbed antibody. (C) The quantitative analysis of Dot-blot result. Two-tailed unpaired Student’s t-test: ^∗∗P < 0.01. (D) Transmission electron micrographs showing capsule thickness in ZY05719 and ΔXress; scale bars, magnification level. The scale bars indicate the magnification size.

FIGURE 7

Model illustrating the proposed regulation of drug resistance and role of Xress-MNTss in S. suis virulence. Under non-stressful conditions, Xress binds directly to MNTss to neutralize the toxicity of MNTss, while Xress and the Xress-MNTss complex bind directly to the promoter region to achieve auto-regulation. Under stressful conditions, the antitoxin is degraded by proteases, MNTss is released, the inhibitory effect of the toxin on zy05719_RS04605 and zy05719_RS04610 is removed. At the same time, MNTss accumulates in the cytoplasm, affecting capsule thickness and biofilm formation with consequent decrease in ZY05719 virulence.