Virulence Regulation Of Two-component System SalK/SalR In Highly Pathogenic Streptococcus Suis Serotype 2

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen associated with a wide range of diseases in pigs, including meningitis, septicaemia, pneumonia, endocarditis and arthritis. Sudden death also may occur as a serious consequence of S. suis 2 infection. Occasionally, S. suis 2 can also be transmitted to human beings through respiratory tract or wound contamination. It was previously thought that SS2 caused only sporadic cases of meningitis and sepsis in humans. However, two major emerging infectious disease outbreaks of SS2 in China (one in Jiangsu Province, 1998, and the other in Sichuan Province, 2005), raised considerable international concerns among the public health professionals. A key feature of these two Chinese outbreaks is the prevalence of streptococcal toxic shock syndrome (STSS) manifesting itself as acute high fever, multiple organ failures, short course of disease and high lethality (62.7%~81.3%).To shed light on the mystery of high virulence of the epidemic outbreak strains of S. suis 2, our joint research group completed a comprehensive study of comparative genomics, decoding the whole genome sequences of two virulent S. suis 2 strains (98HAH12 and 05ZYH33) isolated from Chinese STSS patients. A candidate pathogenicity island (PAI) called 89K was predicted, which is only present in the epidemic strains in these two SS2 outbtreaks but not in other domestic clinical isolates or international virulent strains. However, this bioinformatically predicted candidate PAI needs experimental validation and its linkage to S. suis 2 related high pathogenicity remains unknown.Two-component signal transduction system (TCS) composed of a membrane-bound sensor and a cytoplasmic response regulator, is an important mechanism used by bacteria to sense and response to environmental stimuli. Pathogenic bacteria often use TCSs to control expression of genes encoding bacterial toxins, adhesins, and other virulence-associated molecules that interact with the host and promote survival in vivo. To approach the relationship between the candidate PAI 89K and the high pathogenicity of S. suis 05ZYH33, we focused on a unique TCS named SalK/SalR which is located in 89K to perform further biological function analysis. An isogenic knockout mutant of SalK/SalR was generated and the impact of SalK/SalR deletion on the biological characteristics and virulence was evaluated. Using DNA microarrays and quantitative PCR, the transcription profiles of the wild type strain 05ZYH33 with the SalK/SalR deficient mutant were compared to define the precise regulatory mechanism of SalK/SalR in the virulence of highly pathogenic S. suis 2.In this dissertation, the following experiments are conducted:1. High-efficiency transformation of S. suis 05ZYH33: To construct an efficient electrotransformation method of S. suis 2 virulent strain 05ZYH33, electroporation was performed on gene pulser with E. coli-S. suis shuttle vectors, and transformation efficiencies were evaluated by varying biological and electric parameters of electroporation. By optimizing these factors, an efficient electrotransformation system for S. suis 05ZYH33 was established with a high efficiency of 107 transformants perμg of plasmid pSET2 with a setting of 22.5 kV/cm, 500 ? and 25μF. Additionally, the experimental data show that the spectinomycin resistance gene from pSET2 is a suitable selectable marker for S. suis 2.2. Construction of a knockout mutant of SalK/SalR: The flanking DNA sequences to salKR were amplified from the chromosomal DNA of S. suis 05ZYH33 and were cloned into a pUC18 vector. Then, the SpcR gene cassette amplified from the E. coli-S. suis shuttle vector pSET2 was inserted to generate the salKR knockout vector pUC::salKR. To obtain the isogenic mutantΔsalKR, the competent cells of 05ZYH33 were subjected to electrotransformation with pUC::salKR. For all the SpcR transformants, colony PCR assay was used to examine them with a series of specific primers. The suspected mutant was further confirmed by Southern blotting analysis to ensure that an isogenic knockout mutant of salKR (namelyΔsalKR) was successfully constructed. For complementation analysis, a DNA fragment containing salKR and its upstream promoter was amplified and cloned into the E. coli-S. suis shuttle vector pSET1. Then the recombinant plasmid was electrotransformed into theΔsalKR mutant to screen the complemented strain CΔsalKR with double selection pressure of Spc and Cm and PCR confirmation. 3. Effect of salKR deletion on the general biological characteristics of S. suis 05ZYH33: The general biological characteristics of the wild type strain 05ZYH33 and theΔsalKR mutant were compared under the same conditions. First, the spectinomycin resistance phenotype of the mutant strain was found to be stable inΔsalKR during in vitro culture. Then the growth rate, colony and cell morphology, hemolytic activity and H2O2 sensitivity were examined but, remarkably, no significant differences between the wild type and mutant strain could be ascertained. However,ΔsalKR was found to be less able to resist killing by PMNs, key mediators of innate immunity, when compared with the wild type strain 05ZYH33.4. Impact of salKR mutation on the virulence of S. suis 05ZYH33: To evaluate the impact of salKR deletion on the virulence of 05ZYH33, two sets of experimental infections were carried out in parallel.①Three-week-old piglets (6/group) were challenged intravenously withΔsalKR and the complementary strain CΔsalKR, at a dose of 108 CFU. The virulent wild type strain 05ZYH33 and an avirulent strain 05HAS68 were utilized as positive reference and negative reference, respectively. Results of experimental infections showed that knockout of salKR eliminated the lethality of S. suis 05ZYH33, while functional complementation of salKR into the isogenic mutantΔsalKR restored its high pathogenicity.②Competitive colonization experiment was adopted to analyze the capability of each strain to colonize the various tissues of piglet. Groups of 3 piglets were inoculated intravenously with the wild type strain 05ZYH33,ΔsalKR mutant and a 1:1 mixture of 05ZYH33/ΔsalKR, respectively. Co-colonization experiment results showed that theΔsalKR mutant infected the specific tissues less effectively than the wild type strain, less organs were affected, and lower amounts of mutant bacteria were re-isolated from these organs. However, theΔsalKR mutant could not colonize any susceptible tissue of piglets when administered alone.5. Role of SalK/SalR in the virulence regulation of S. suis 05ZYH33: To gain further insights into the network mediated by SalK/SalR, whole-genome DNA microarray (CombiMatrix) was applied to reveal the differential transcription profiles between the wild type strain 05ZYH33 with theΔsalKR mutant. The microarray data were then confirmed by quantitative PCR independently. In total, the absence of SalK/SalR led to decreased expression of 26 genes spread throughout the genome, which can be roughly categorized into the following 3 groups: (i)Genes involved in substance transport and metabolism; (ii)genes involved in recombination/repair and transcription; (iii)Genes encoding membrane proteins and some proteins of unknown function. Among these down-regulated genes, two potential virulence factors were identified, which encode orotidine-5-phosphate decarboxylase and phosphotransferase system, respectively.In conclusion, a unique two-component regulatory system SalK/SalR has been identified from a putative 89K PAI in Chinese isolates of highly pathogenic S. suis 2. Prior to this study, SalK/SalR is only known to be associated with the production of a kind of lantibiotic peptide named salivaricin A (SalA) in S. salivarius, as yet, this TCS has not been linked to the regulation of bacterial virulence. Our data confirm, for the first time, that SalK/SalR is absolutely indispensable for the full virulence of Chinese highly pathogenic S. suis 2 strains. Not only does this investigation provide experimental evidence for the validity of the candidate 89K PAI, but it adds novel insights into the infectious disease pathogenesis of S. suis 2.