Study On The Regulation Of Pathogenicity By Two-component Regulatory Systems 1910HK/RR In Streptococcus Suis Serotype 2

Streptococcus suis serotype 2 (SS2) is one of the most important swine pathogens, and an emerging, life-threatening zoonotic agent in both pigs and humans. Based on the CPS antigens of S. suis,35 serotypes have been characterized. SS2 is most commonly and most virulent of these serotypes. SS2 cause great economic loss to the pig industry worldwide. Due to high economic losses as well as threat to human life, the study of SS2 pathogenesis, and the development of new vaccines is crucial for prevention and control of this disease.Development of the disease requires temporal and coordinated expression of a series of genes that allow the prospective pathogen to shift to its pathogenic state and adapt to a hostile environment in the host. TCS control the expression of virulence factors in a wide range of bacterial species in response to external stimuli. For bacterial pathogens, they have evolved virulence factors and regulation systems that allow adaptation to different environments within a host ensuring their survival. TCS generally consist of a sensor kinase and response regulator and are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Many results have implicated these systems as playing an important role in the regulation of a variety of essential processes including cell-cycle progression, pathogenicity, and development.Two SS2 strains 05ZYH33 and 98HAH12 isolated in China have been sequenced. The results predict that there are 15 TCS in both the strains together with single, unpaired response regulator. To date, only the SalK/SalR system and two orphan response regulators, RevS and CovR.In this work, we constructed a mutant strain of the TCS 1910HK/RR, designatedΔ1910hk/rr and measured its virulence in vitro and in vivo. So the following researches were explored.1. Construction and verification of SS2 mutant strain (Δ1910hk/rr) and complementation strain (CΔ1910hk/rr)The upstream and downstream flanking regions of 1910hk/rr were separately amplified fromSS 2 wild-type strain SC19 genomic (WT) DNA respectively. Followed by digestion with the corresponding restriction enzymes, the PCR products were directly cloned into a pSET4s vector. Then, the plasmid, pSET4s-1910hk/rr was electroporated into WT, and the resultant strains were grown at 28℃in the presence of spectinomycin (100μg/mL) selection and subsequently passaged at 37℃in the absence of spectinomycin selection as described previously. Successful deletion of the 1910hk/rr was confirmed by PCR and RT-PCR. For complementation analysis, a DNA fragment containing 1910hk/rr and its upstream promoter were amplified and cloned into the E.coli-S.suis shuttle vector pAT18. Then the recombinant plasmids were electrotransformed into the mutants to screen the complemented strains CΔ1910hk/rr.2. Biology characteristic research of the SS2 mutant strainΔ1910hk/rr, complementation strain CΔ1910hk/rr and SS2 wild type srain (WT)The results of the growth curve ofΔ1910hk/rr,CΔ1910hk/rr and WT showed thatΔ1910hk/rr grow slower than that of WT. The hemolytic activity of wild-type strain was more high higher than that of mutant strain. However, there were not significant difference-betweenΔ1910hk/rr and WT. The hemolytic activity of complementation strain was restored, and was about two times higher thanΔ1910hk/rr. But it did not reach the level of WT. To assess whether the lack of 1910HK/RR affected the cellular adhesion of SS2, the adherence efficiencies of the WT and theΔ1910hk/rr mutant to human continuous laryngeal epithelial cell line Hep-2 cells were compared. The results showed that WT showed significantly more adhesion to both Hep-2 cells than theΔ1910hk/rr, indicating the role of 1910HK/RR as an important mediator in the cellular-adhesion process. LD50 values were 4.14×107CFU for WT,1.22×109CFU forΔ1910hk/rr. Compared with parent strain,Δ1910hk/rr was attenuated 29.5 fold. The effect of 1910HK/RR on PMN killing was further examined. PMN were incubated with two strains for two hour and the bactericidal activity of PMN cells was determined. Remained bacteria were decreased in theΔ1910hk/rr group. In contrast, the WT strain still multiply when incubated with PMN, indicating that 1910HK/RR is able to enhance the bactericidal activity of PMN.3. Impact of the 1910HK/RR on the virulence of SS2 WTIn order to evaluate whether the 1910HK/RR contributes to the virulence of SS2 in vivo, an experimental infection of CD-1 mice was performed. Morbidity and mortality analysis of CD-1 mice was performed to study pathogenesis of 1910HK/RR. Five-week-old CD-1 mice were intraperitoneally inoculated with 108 CFU/mL bacteria and the survival was monitored over a 12-day period. The results showed that almost all animals in the WT group presented severe clinical signs of sepsis, such as depression, rough hair coat, swollen eyes, weakness and prostration during the first three days post-infection. Seven mice died from septicemia in the WT group during 48 hour post-inoculation. In contrast, only one mice died in theΔ1910hk/rr group during the 48 hours post-inoculation and all of remaining nine mice did not present severe clinical signs of sepsis associated with SS2 infection during the trial, recovering after 4 days post-inoculation. In order to further confirm the clinical signs ofΔ1910hk/rr mutant in vivo, bacteria loads of organs were monitored. According to results above, mice were intraperitoneally inoculated with either the strainΔ1910hk/rr or WT at a dose of 1×107 CFU. Results showed that bacteria loads in blood following inoculation were lower in theΔ1910hk/rr group, suggesting that theΔ1910hk/rr could not multiply effectively in organs compared with WT.The contribution of 1910HK/RR to SS2 virulence was assessed by infecting piglets with theΔ1910hk/rr mutant or WT strain. All six piglets inoculated intranasally with WT strain at the dose of 1×105 CFU developed hyperthermia and depression within 48 hours, including two of which that died withing 24 hours. Later, most of the typical disease symptoms, including limping, swollen joints, shivering, central nervous system failure and respiratory failure were observed. Four of six piglets (4/6) infected with WT strain died within 48 h post-infection. In contrast, none of the piglets infected with theΔ1910hk/rr developed any of the clinical signs mentioned above within the first 24 hours with exception of one which was slightly depressed. However, one piglet died within 48 hours. The remaining five recovered from fever showing none of other typical disease symptoms and remained healthy throughout the 21 day experiment. To better evaluate the virulence attenuation ofΔ1910hk/rr, colonization experiments and histopathological examinations were carried out. The results showed that almost all the bacterial counts recovered from different tissues ofΔ1910hk/rr mutant infected-piglets were significantly lowercompared with WT infected-piglets. Notably, the bacterial counts from theΔ1910hk/rr infected group was lower than WT. Histopathological examination of brain and lung tissues demonstrated that the blood vessels of brain from the WT infected group were filled with numerous macrophages and neutrophils compared to theΔ1910hk/rr infected group. In fact, the presence of high numbers of leukocytes suggests that the exacerbated inflammatory response might be the cause of death within the first 48 hours.4. Role of 1910HK/RR in the virulence regulation of WTTo gain further insights into the network mediated by 1910HK/RR, DNA microassay, quantitative PCR was applied to reveal the differential transcription profiles between the strain WT with theΔ1910hk/rr independently. In this study we used a microarry which was made by using available the SS2 05ZYH33 strain whole genome sequence. To compare with the wild type SS2 strain SC19 gene expression,Δ1910hk/rr shows that 105 genes were down-regulated 1.5-fold and 114 genes were upregulated 1.5-fold. In total, the absence of 1910hk/rr led to changed expression of over 200 genes. Of the 114 upregulated genes,24 genes were investigated. They can be roughly categorized into the following four groups:(Ⅰ) Genes involved in ABC-type amino acid transport and metabolism; (Ⅱ) Genes involved in metabolism; (Ⅲ) Genes encoding membrane proteins and some proteins of unknown function, associted with virulence. (Ⅳ) Genes encoding sialic acid synthesis gene. The intereaction of DNA promoter with regulator 1910RR was confirmed by EMSA.