SalK/SalR, a two-component signal transduction system, is essential for full virulence of highly invasive Streptococcus suis serotype 2

Abstract

Background: Streptococcus suis serotype 2 (S. suis 2, SS2) has evolved into a highly infectious entity, which caused the two recent large-scale outbreaks of human SS2 epidemic in China, and is characterized by a toxic shock-like syndrome. However, the molecular pathogenesis of this new emerging pathogen is still poorly understood.

Methodology/principal findings: 89K is a newly predicted pathogenicity island (PAI) which is specific to Chinese epidemic strains isolated from these two SS2 outbreaks. Further bioinformatics analysis revealed a unique two-component signal transduction system (TCSTS) located in the candidate 89K PAI, which is orthologous to the SalK/SalR regulatory system of Streptococcus salivarius. Knockout of salKR eliminated the lethality of SS2 in experimental infection of piglets. Functional complementation of salKR into the isogenic mutant DeltasalKR restored its soaring pathogenicity. Colonization experiments showed that the DeltasalKR mutant could not colonize any susceptible tissue of piglets when administered alone. Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased. Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the DeltasalKR mutant.

Conclusions/significance: These findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI.

Figure 1. Identification and characterization of a unique two-component regulatory system SalK/SalR in the putative 89K PAI.

A), Discovery of a unique two-component signal transduction system (TCSTS), SalK/SalR from 89K island. B), Aberrant average GC content of salKR which is much less than that of 89K and the whole genome. C), Phylogenetic analysis of the response regulator SalR and the sensor histidine kinase SalK of SalK/SalR regulatory system, with some related members known at the level of amino acids.

Figure 2. Transcriptional analysis of the salKR locus and its flanking genes of S. suis 05ZYH33.

A), Genomic organization of the salKR locus and its flanking genes in S. suis 05ZYH33. Arrows represent the length and direction of transcription of the genes surrounding salKR. The putative transcripts initiated at P05SSU0942 and PsalK are depicted by thin horizontal arrows. Stem-loop structures represent relevant potential transcriptional terminators. The location of the primer pairs used in RT-PCR analysis are indicated by inverted arrowheads. B), RT-PCR analysis of the salKR locus and its flanking genes of 05ZYH33. Total RNA extracted from mid-exponential-phase cultures, and genomic DNA were analysed by RT-PCR and PCR, respectively, using primer pairs P1/P2 (lane 1), P3/P4 (lane 2), P5/P6 (lane 3), P7/P8 (lane 4), P9/P10 (lane 5), P11/P12 (lane 6), P13/P14 (lane 7) and P15/P16 (lane 8). The 1 kb DNA ladder marker is shown to the left (M).

Figure 3. Construction and confirmation analysis of the knockout mutant strain ΔsalKR.

A), Strategy for deletion mutagenesis of salKR in S. suis 05ZYH33 by allelic replacement with a spectinomycin resistance cassette and schematic representation of the chromosomal structures before (left) and after (right) double cross-over (I) and single cross-over (II and III) recombination events between pUC::salKR and the chromosome of S. suis 05ZYH33. The location of the primers used in multiple-PCR detection are indicated by inverted arrowheads. The dotted lines represent the chromosomal sequences flanking the left and right arms of the construct. B, C and D), Southern hybridization analysis of the salKR region of S. suis wild type strain 05ZYH33 (lane 1), ΔsalKR mutant (lane 2), and a 3′ single cross-over mutant with pUC::salKR integrated into the chromosome of 05ZYH33 (lane 3). Genomic DNA from each strain was digested with Cla I and separated on 0.7% agarose gel. The hybridization probes used were as follows: Spc^R gene (B), an internal fragment of salKR (C), and pUC18 (D). E), Multiple-PCR analysis of the ΔsalKR mutant. The primer combinations used in PCR are presented upon the lanes. Genomic DNA from the following strains were used as templates: wild type strain 05ZYH33 (lane 1, 4, 7, 10, 13, 16, 19 and 22), ΔsalKR mutant (lane 2, 5, 8, 11, 14, 17, 20 and 23) and the 3′ single cross-over mutant (lane 3, 6, 9, 12, 15, 18, 21 and 24). The 1 kb DNA ladder marker is shown to the left (M). Theoretical size (bp) of each of the PCR products generated with the primer combinations was shown in Table S2.

Figure 4. Cell morphology and ultrastructure of S. suis wild type strain 05ZYH33 and mutant strain ΔsalKR.

A), Morphology of bacteria under the light microscope using India ink staining (×1000). B), Transmission electron micrographs of bacteria. The capsule is highlighted by black arrows. The bar indicates the magnification size.

Figure 5. SPF-piglet experimental infections.

Groups of 6 piglets were challenged intravenously with S. suis wild type strain 05ZYH33, mutant strain ΔsalKR, complemented strain CΔsalKR, and avirulent strain 05HAS68, respectively, at a dose of 10⁸ CFU/piglet. Survival time (days) of individual piglets is indicated.

Figure 6. Decreased resistance of ΔsalKR to PMN-mediated killing.

SS2 wild type strain 05ZYH33 and mutant strain ΔsalKR were co-incubated respectively with pig neutrophils (PMNs) at a moi of 10∶1. At each time, PMNs were lysed and bacteria were plated on growth agar. Colonies were enumerated the next day, and percent bacteria killed was calculated. Data are expressed as the mean±SD of three independent experiments. *, P<0.05.