| Streptococcus suis serotype2(S. suis2) is an important swine pathogen, causing arthritis, endocarditis, meningitis, pneumonia, and septicemia. It is also a major zoonotic agent that can cause life-threatening infections in humans. Two recent large-scale outbreaks of S. suis2in China were associated with streptococcal toxic shock syndrome, manifesting as acute high fever, multiple organ failure, short course of disease, and high lethality. These outbreaks were of major concern for global public health. Despite much research on S. suis2, little is known about the underlying mechanism that this emerging organism has evolved to enhance its pathogenicity.Two-component signal transduction systems (TCSTS) are commonly used by bacteria to influence the transcription of specific genes or operons, and to mediate cellular changes in response to environmental stimuli. Recent results have implicated critical roles for these systems in the regulation of a variety of essential processes, including cell-cycle progression, pathogenicity, and developmental pathways. Nisin Z has been reported to be positively autoregulated by extracellular nisin via the NisK/NisR system in Lactococcus lactis. The BceR/BceS TCSTS, comprising a four-component system BceRS-BceAB, induces the expression of the ABC transporter, which in turn removes the antibiotic from its site of action, mediating the resistance of the cell. However, these two systems have not previously been reported in S. suis2.Analysis of89K identified two TCSTS. One of which, called SalK/SalR, proved to be essential for full virulence of the highly pathogenic S. suis serotype2isolate, while the other appeared to be orthologous to the NisK/NisR system of Lactococcus lactis. To date, CiaRH, Ihk/Irr, VirR/VirS, and two orphan response regulators, RevS and CovR, have been described, all of which have been implicated in the virulence of S. suis2. Thus, my research has been focused on NisK/NisR and BceR/BceS. The goal is to elucidate the virulence impact of TCSTS and to identify genes regulated by TCSTS. Major contents are as the following:1. Construction and identification of the△nisKR and△bceRS mutantsFirst, primers were designed based on the whole genome sequences of05ZYH33using the genome of S. suis SC19as the template and thermosensitive suicide vector pSET4s was used as shuttle vector. The knockout mutant was constructed by homologous recombination and the double-crossover event was confirmed by PCR and Southern blot.2. Biological characteristics of the△nisKR and AbceRS mutants The growth and hemolytic activity of the mutants were studied. It was found that the growth of the mutant strain in TSB containing10%newborn bovine serum showed no significant difference to that of the wild-type. However, the hemolytic titer of the mutants was more than twofold lower than that of wild-type. Cells and mice models were used to assess the contribution of NisKR and BceRS to the virulence of S. suis2. The results demonstrated that the AnisKR and AbceRS mutants displayed significantly less adherence (a85.4%and53.05%decrease in adherence respectively) to HeLa cells compared with the wild-type. Moreover, the invasion capacity of the AnisKR and AbceRS mutant was significantly reduced (a90.9%and76.77%decrease in invasion). Besides, it was found that disruption of NisKR and BceRS decreases resistance of this pathogen to killing by PMNs and render S. suis2more vulnerable to both phagocytosis and bactericidal activity of RAW264.7cells. For virulence studies, BALB/c mice were challenged intraperitoneally with1ml of WT or AnisKR S. suis at approximately8×108CFU/ml in TSB. The results showed that almost all mice in the wild-type group presented severer clinical signs of infection and significantly higher mortality compared with the mice challenged with the mutant. To better evaluate the virulence attenuation of the mutant, colonization experiments were performed. BALB/c mice were intravenously injected with WT and AnisKR at a dose of5×107CFU in TSB. The results indicated that bacterial counts recovered from heart, lung, brain, and blood of△nisKR-infected mice were lower than those of wild-type-infected mice at each time point. All of the results strongly suggested the importance of the NisK/NisR and BceR/BceS systems in the pathogenesis of S. suis2.3. Primary study on the regulation mechanism of the NisK/NisR and BceR/BceS systems in S. suis2Genes differentially expressed between the wild type and the mutant were identified by the method of RNA-SEQ. The results showed that22genes were down-regulated and7genes were up-regulated by2-fold in AnisKR;4genes were down-regulated and15genes were up-regulated by2-fold in AbceRS. Meanwhile, the NisR and BceR were successfully expressed and purified. Then we optimized the first use of an in vitro genome-wide method (DAP-seq) to map response regulator binding sites in S. suis2and get a better understanding of its regulation mechanism. |
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