The orphan response regulator CovR: a globally negative modulator of virulence in Streptococcus suis serotype 2

Abstract

Streptococcus suis serotype 2 is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans. However, the pathogenesis of S. suis serotype 2 infection is not well understood. In this study, we report that an orphan response regulator, CovR, globally regulates gene expression and negatively controls the virulence of S. suis 05ZYH33, a streptococcal toxic shock syndrome (STSS)-causing strain. A covR-defective (DeltacovR) mutant of 05ZYH33 displayed dramatic phenotypic changes, such as formation of longer chains, production of thicker capsules, and increased hemolytic activity. Adherence of the DeltacovR mutant to epithelial cells was greatly increased, and its resistance to phagocytosis and killing by neutrophils and monocytes was also significantly enhanced. More importantly, inactivation of covR increased the lethality of S. suis serotype 2 in experimental infection of piglets, and this phenotype was restored by covR complementation. Colonization experiments also showed that the DeltacovR mutant exhibited an increased ability to colonize susceptible tissues of piglets. The pleiotropic phenotype of the DeltacovR mutant is in full agreement with the large number of genes controlled by CovR as revealed by transcription profile analysis: 2 genes are positively regulated, and 193 are repressed, including many that encode known or putative virulence factors. These findings suggested that CovR is a global repressor in virulence regulation of STSS-causing S. suis serotype 2.

FIG. 1.

Construction of an isogenic covR mutant of S. suis 05ZYH33. (A) Diagram of the covR locus from S. suis 05ZYH33 and strategy for insertional inactivation of covR. The spectinomycin resistance gene, indicated by a thick black arrow, was inserted in the unique XhoI site of covR. The thin arrows indicate the primer positions used for the construction and identification of the covR knockout mutant (ΔcovR). (B) Confirmatory PCRs of the ΔcovR mutant. The primer pairs used in the PCR analysis are indicated above the lanes. Genomic DNAs from the mutant ΔcovR strain (lanes 1, 3, 5, and 7) and the wild-type strain, 05ZYH33 (lanes 2, 4, 6, and 8), were used as templates. The DNA molecular size marker is a 1-kb DNA ladder (lane M). (C) RT-PCR analysis of covR gene transcripts. Total RNAs were extracted from independent S. suis serotype 2 cultures of the wild type (lane 1), the ΔcovR mutant (lane 2), and the complemented strain, CΔcovR (lane 3). cDNAs generated from these RNA samples were subjected to RT-PCR analysis with covR-specific primers (CovR-F and CovR-R). The RT-PCR products were analyzed by electrophoresis on a 1.0% agarose gel (lanes 1, 05ZYH33; lanes 2, ΔcovR strain; lanes 3, CΔcovR). The 1-kbp DNA ladder marker is shown on the right (lane M).

FIG. 2.

Comparison of the in vitro growth rates of the wild-type strain, 05ZYH33, and the ΔcovR mutant. (A) Bacteria were cultured in THB at 37°C. (B) Bacteria were cultured in THB containing 10% FBS at 37°C. The results shown are representative of three independent experiments. OD₆₀₀, optical density at 600 nm.

FIG. 3.

Cell morphology of the S. suis wild-type strain, 05ZYH33, and the mutant strain, ΔcovR. (A) Light microscope morphology of S. suis strains using Gram staining (magnification, ×1,000). (B) Transmission electron micrographs of bacteria cultured in THB containing 10% FBS. The capsule is indicated by the arrows.

FIG. 4.

Hemolytic-activity analysis of the ΔcovR mutant and its parental strain, 05ZYH33. (A) Hemolytic activities of S. suis strains streaked on THB agar plates containing 6% sheep blood and incubated for 48 h at 37°C. (B) Titration of hemolytic activities of S. suis serotype 2 culture supernatants. The horizontal line indicates the highest dilution of hemolysin that induced at least 50% lysis of erythrocytes. OD₅₄₀, optical density at 540 nm.

FIG. 5.

Hyperadhesion of the ΔcovR mutant to human epithelial (Hep-2) cells and endothelial cells (HUVEC). The results were determined after a 1-h coincubation of various S. suis strains with Hep-2 cells and HUVEC at a multiplicity of infection of 10, followed by extensive washing of nonadherent bacteria and cell lysis to retrieve 100-μl aliquots of total cell-associated bacteria for viable plate counts. The results shown are the means ± standard deviations of three independent experiments. *, P < 0.01.

FIG. 6.

Enhanced resistance of the ΔcovR strain to phagocytosis and killing by neutrophils and MONOs. The wild-type and mutant strains were incubated with PMNs or MONOs at a bacteria-to-cell ratio of 10:1, respectively. The cells were lysed after 1 h of incubation, and the survival percentage of each strain was calculated as follows: (CFU_PMN+/CFU_PMN−) or (CFU_MONO+/CFU_MONO−) × 100%. The data are expressed as the means ± standard deviations of three independent experiments. *, P < 0.05.

FIG. 7.

Pig infection experiments. Groups of six SPF piglets were challenged intravenously with approximately 10⁸ CFU of the indicated strains. The survival time for each piglet is indicated. Each datum point represents one piglet.

FIG. 8.

Pathological examination of infected piglets. Kidney and heart tissues from the autopsy specimens of piglets infected by the wild-type strain, 05ZYH33, and the ΔcovR mutant were prepared for light microscopy and TEM observation in order to examine the differences in the pathological changes produced by the two strains.

FIG. 9.

Differential colonization abilities of the wild-type and ΔcovR mutant strains in susceptible tissues of piglets. The ΔcovR mutant was mixed with the wild type at a ratio of 1:1 and inoculated intravenously into six SPF piglets. When typical S. suis serotype 2 infection symptoms developed, the piglets were sacrificed, the bacteria recovered from various tissues were enumerated, and the percentage of each strain within the population was calculated. The data are expressed as the means and standard deviations of three independent experiments. *, P < 0.05.

FIG. 10.

Correlation of DNA microarray and real-time PCR results. The changes in the relative gene transcription (ΔcovR to 05ZYH33) of nine selected genes obtained by DNA microarray and real-time PCR analyses were log₂ transformed, and the values were plotted against each other to evaluate their correlation.