Research On Two-component Signal Transduction System 2148hk/rr Of Streptococcus Suis Serotype 2

Streptococcus suis is a kind of Gram-positive facultative anaerobes, including 35 serotypes (1-34 type and 1/2), which S. suis serotype 2 (SS2), is the most widespread and pathogenic. Human infection with S. suis is characterized by purulent meningitis, endocarditis, associated with deafness, movement disorders, sepsis and other symptoms. Notably, two recent large-scale outbreaks of human streptococcus toxic shock syndrome (STSS)caused by SS2 in China in 1998 and in 2005 have posed public health concerns worldwide. S. suis , especially S. suis in China is not well characterized up till now. It's lack of overall understanding about the mechanisms involved in the pathogenesis and virulence of S. suis. Although some virulence factors were identified in association with pathogenesis, such as capsular polysaccharide(CPS) involved in resistance phagocytosis, muramidase-released protein(MRP), extracellular protein factor(EF), suilysin(SLY), arginine deiminase(ADiS) and so on. Currently, the researches mainly focus on identification of S. suis novel virulence factors.However, identification of single virulence factor can't account for pathogenic mechanism of SS2's outbreak and epidemic in China. It's more important to investigate the regulatory systems which regulate the function of virulence genes. Two-component signal transduction systems (TCSTS) is a mechanism used by bacteria to adapt to and survive in the changing environment. Pathogenic bacteria often use TCSTSs to regulate the expression of genes encoding bacterial toxins, adhesins, and other virulence-associated molecules that interact with the host and promote survival in vivo.In general, a TCSTS consists of a membrane sensor (histidine kinase, HK) and a cytoplasmic response regulator (RR), which is generally a DNA-binding protein that modulates the expression of certain target genes. In S. suis serotype 2, at least 13 TCSTSs and 2 orphan response regulators have been identified. It is known that bacterial virulence is tightly regulated by both positive and negative feedback mechanisms. Therefore, environmental regulation of virulence gene expression in S. suis serotype 2 via TCSTSs deserves to be further addressed. In the genome of S. suis 05ZYH33, the peptides encoded by SSU05₂148 and SSU05₂149 exhibit 30% amino acid sequence identity with the VirR/S TCSTS of Clostridium perfringens, and hence, the TCSTS (encoded by SSU05₂148 and SSU05₂149) was renamed 2148hk/rr.Prior to evaluating the effect of 2148hk/rr inactivation on the virulence of S. suis 05ZYH33 in vivo, we first examined the growth characteristics of the mutant strainâ–³2148hk/rr mutant in vitro. A slight difference in growth of the wild-type and mutant strains was reproducibly observed. Theâ–³2148hk/rr mutant grew at a lower rate than the parent strain 05ZYH33 in Todd-Hewitt Broth(THB). Adherence of pathogenic bacteria to the mucosal surface is considered to be an essential step in the infectious process. The mutant strainâ–³2148hk/rr exhibited a significantly decrease in adherence to epithelial cells Hep-2 (P<0.05) than the wild-type strain. In susceptibility to whole blood experiment, the mutantâ–³2148hk/rr to resist the killing of innate immune cells in human whole blood was also weakened, and easier to be cleared by the host. CD1 mice competitive infection experiment showed that the proliferation of the mutant strainâ–³2148hk/rr was inhibited by the wild-type strain. When the mutantâ–³2148hk/rr and wild-type strain were administered at the same time, the piglet competitive infection index(CI) value of the mutant was zero, indicating the mutant strain was more easily removed compared to the wild-type strain. The results suggest that the TCSTS 2148hk/rr may regulate some virulence factors at the transcriptional level.To identify TCSTS 2148hk/rr regulated genes and also putative virulence associated transcriptional alterations in theâ–³2148hk/rr mutant, a DNA microarray based comparative-transcriptomics approach was applied. In total, the inactivation of 2148hk/rr led to 426 differentially expressed genes spread throughout the genome (19.42%), with a twofold cutoff. Of these, 256 genes were 2148hk/rr repressed and 170 genes were positively regulated by 2148hk/rr. The Capsule (CPS) biosynthesis locus of S.suis serotype 2 consists of 14 open reading frames (SSU05₀564-0577). And transcripts of CPS clusters were at a low level in the absence of 2148hk/rr, consistent with the phenotypic changes of theâ–³2148hk/rr mutant, and production of thinner capsules. The regulated genes can be classified into several broad categories such as carbohydrate, amino acid, nucleotide transport and metabolism, DNA replication, recombination and repair and so on. The transcripts of other TCSTSs, SalK/R, CiaR/H, YycF/G, 1596hk/rr, 0884hk/rr were also at a low level in the mutant strainâ–³2148hk/rr. The results indicated that these TCSTSs were positively regulated by TCSTS 2148hk/rr.In order to independently confirm the microarray data, the relative transcript levels of 45 selected genes were measured by quantitative real-time PCR analysis. These genes were chosen because the majority of them were identified as encoding factors influencing bacterium-host interactions. These genes identified as differentially expressed by microarray analysis were consequently confirmed to exhibit altered transcript levels by real-time PCR assays. There was a strong positive correlation (R2 = 0.926) between the data obtained by these two different techniques. The virulence factor (SSU05₁403) and 6 putative operons (SSU05₀564-0577, SSU05₀578-0581, SSU05₀624-0628, SSU05₁094-1095, SSU05₁216-1221, SSU05₁778-1780) were analyzed by electrophoretic mobility shift assay (EMSA). The result showed that these target genes were regulated by 2148hk/rr in an indirect way.To gain further insights into the network/circuit of the TCSTSs, whole-genome DNA microarray was applied to reveal the differential transcription profiles between other TCSTSs mutant (â–³SalK/R,â–³CovR,â–³RevS,â–³CiaR/H,â–³LuxS,â–³1910hk/rr,â–³1661hk/rr)and wild-type strain (05ZYH33, SC19). All data on transcriptional changes of 2,194 genes anchoring on 05ZYH33 genome microarray was collected and the cluster analysis was performed based on gene classes and COG categories. The transcription of genes encoding proven or predicted virulence factors was found to be regulated in different TCSTSs, which are helpful to understand the regulatory network of virulence factor in 05ZYH33. TCSTS 2148hk/rr plays an important role in regulating the CPS clusters, and the other tested TCSTSs just regulate a few genes of CPS clusters without phenotypic changes. The genes regulated by TCSTS CiaR/H and TCSTS 1910hk/rr have a similar tendency and amplitude in the categories of carbohydrate, amino acid transport and metabolism, and cell envelope biogenesis. The cluster analysis also showed that TCSTS CiaR/H was a global repressor regulatory system for 89K PAI. Additionally, the gene sly (SSU05₁403) encoding suilysin was up-regulated in the TCSTSs mutants includingâ–³SalK/R,â–³CiaR/H,â–³CovR, andâ–³2148hk/rr. Deletion of SalK/R or 2148hk/rr lead to elimination of the lethality, but CovR mutant is more lethal than the wild-type strain. It indicates that the higher transcriptional level of sly does not mean the more virulence of the mutants.