| Streptococcus suis serotype 2 (S. suis 2) has been regarded as an important zoonotic pathogen in the world and is responsible for a variety of life-threatening infections in humans and pigs, including meningitis, arthritis, septicaemia and pneumonia. It was previously thought that S. suis 2 caused only sporadic cases of meningitis and sepsis in humans. However, two recent large-scale outbreaks caused by S. suis 2 in China in 1998 and 2005 have provoked considerable public health concerns in the worldwide. These two outbreaks showed a prevalent feature of streptococcal toxic shock syndrome (STSS), manifested as acute high fever manifesting itself as acute multiple organ failures, high fever, short course of disease and high lethality.In general, STSS was initially reported to be a sequel of Streptococcus pyogenes (GAS). To date, superantigens have been correlated with STSS. Although a series of virulence determinants of S. suis 2 have been identified, such as capsule polysaccharide (CPS), muramidase-related protein (MRP) and suilysin, no superantigen or homologue responsible for STSS was found in S. suis 2, suggesting an alternate mechanism for its virulence.Previously, our joint research group identified an unique pathogenicity island (PAI) designated 89K which is specific to the STSS-causing epidemic strains of S. suis 2. Further bioinformatics analysis of the 89K PAI identified a type IV-like secretion system (T4SS-like system) located on the 5’half of the 89K island. T4SS contributes to evolution of pathogens by mediating horizontal gene transfer and delivering pathogenic effectors to host cells, thus, contributing to the virulence of bacteria. Additionally, examples of T4SS involvement in the modulation of cytokine secretion or inflammatory response were previously observed in several Gram-negative bacteria. Ye et al. recently reported that serum levels of cytokines were more elevated in patients with STSS caused by S. suis 2 than in those with meningitis only. This suggests that the highly invasive S. suis 2 strains adopt certain incentive mechanisms that trigger a proinflammatory cytokine cascade that leads to excessive systemic inflammatory responses. It is known that there is a close link between excessive release of endogenous inflammatory mediators and the development of STSS. This implies that the T4SS-like system harbored within the 89K PAI may be responsible for the pathogenesis of STSS caused by S. suis 2. In this study, we knocked out the two key components (VirD4-89K and VirB4-89K) of the T4SS-like system and assessed the contribution of this system to the development of STSS induced by the highly invasive S. suis 2.In this dissertation, the following experiments are conducted:1. Construction of knockout mutants of T4SS-like system: Based on the bioinformatics analysis, a T4SS-like system was identified at the 5’-half of the 89K PAI. To test the role of the T4SS-like system in the pathogenesis of S. suis 2, and its contribution to the development of STSS, knockout mutants of two key components (VirD4-89K and VirB4-89K) of the T4SS-like system were constructed by double cross-over recombination through allelic replacement with a spectinomycin (spc) resistance gene cassette. These knockout mutants were designated△virD4-89K and△virB4-89K, respectively.2. Role of the T4SS-like system in virulence of S. suis 2: Prior to evaluating the virulence of△virD4-89K and△virB4-89K, the basic biological characteristics of these two mutants were examined. Compared to the wild-type strain 05ZYH33, knockout mutants did not exhibit noticeable differences in growth rate, length of the chain, thickness of the capsular material and hemolytic activity (data not shown). To investigate the role of the T4SS-like system in the pathogenesis of the highly pathogenic strain 05ZYH33, a fatal infection model in BALB/c mice was designed to compare the virulence of the wild-type strain 05ZYH33, mutant△virD4-89K or△virB4-89K, and the control strain P1/7. All of the mice challenged with the wild-type strain developed typical clinical symptoms of S. suis 2 infection, while mice infected with mutant△virD4-89K or△virB4-89K showed mild or no clinical signs. 100% of those challenged with the wild-type strain had succumbed to fatal infection within the first 24 h (post infection, p.i.), whereas the group of mice inoculated with either△virD4-89K or△virB4-89K were shown to have 60% survival rates at the experimental end. A significant difference (P = 0.006, for△virD4-89K; P = 0.003, for△virB4-89K) was observed between the wild-type infected group and mutant-infected group.3. Systemic measurement of proinflammatory cytokines in BALB/c mice: To investigate the effect of the T4SS-like system on excessive cytokine release associated with STSS caused by the highly invasive S. suis 2, cytokine production in BALB/c mice infected with the wild-type strain 05ZYH33, mutant (△virD4-89K or△virB4-89K), the control strain P1/7 were compared. The results showed that serum levels of TNF-α, IL-6 and IL-12p70 was significantly lower in mice infected with either△virD4-89K or△virB4-89K than in mice infected with the wild-type strain, particularly at 8 h p.i.. Inflammatory cell infiltration was observed in the liver tissue of the wild type-infected group, but no or few inflammatory cells were seen in both mutant groups (△virD4-89K or△virB4-89K). These results indicate that△virB4-89K and△virD4-89K have a decreased ability to elicit a host response to induce excessive cytokine production.4. Systemic measurement of proinflammatory cytokines in cell model: In parallel with the in vivo experiments, efforts were done to observe cytokine induction in monocytes stimulated with the four indicated strains (the wild-type strain 05ZYH33, mutants△virD4-89K or△virB4-89K, and the reference strain P1/7). All of the analyzed strains led to a time-dependent production of cytokines in monocytes, suggesting that the cell model is reliable. Unexpectedly, no statistical differences of cytokine production profiles were observed when monocytes stimulated with these different strains. Considering the fact that many other cell types, including T-cells, B-cells, natural killer (NK) cells, and neutrophils, also produce inflammatory cytokines, fresh whole blood samples were therefore used for measurement of cytokine response. Once again, we did not get expected results. No significant differences were found in the kinetic patterns of TNF-αand IL-6 production induced by the four strains.Conclusion:1. Knockout mutants△virB4-89K and virD4-89K were generated.2. Deletion of either△virB4-89K or virD4-89 leads to significant elimination of the lethality of the wild type stain, 05ZYH33.3. Knockout of the two key components (VirD4-89K and VirB4-89K) of the T4SS-like system impaired its ability to trigger host immune response in experimental infection of mice.4. Results confirmed that the T4SS-like system contributes to the development of STSS caused by the highly invasive S. suis 2. The T4SS-like system induces transfer of some pathogenic effectors that can elicit excessive systemic inflammatory responses in mice. |
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