| Aim:The gram-negative bacterium Yersinia pestis is the causative agent of bubonic, septicemic and pneumonic plague, a highly lethal, zoonotic disease to humans. The thorough analysis of the pathogenic mechanism of Y. pestis is the most effective way to control the epidemic of plague. Quorum sensing (QS) is a cell-density dependent global regulation system, which is used for cell to cell communication mediated by the autoinducer. The aim of the research is to reveal the relationship between the QS system and the survival and virulence of Y. pestis.Methods:Agrobacterium tumefaciens NTL4(pZLR4) and Vibrio harveyi BB170 can be used as the reporter to analyze the existence of acyl-homoserine lactone (which is the autoinducer of type-I QS system, abbreviated as AHL) and furanosyl borate diester (which is the autoinducer of type-II QS system, abbreviated as AI-II) respectively. In the research work, firstly, NTL4 and BB170 were used to analyze the autoinducer-producing property of Y. pestis qualitatively. And then the HPLC-MS-MS system was used to identify the AHLs that were produced by two kinds of AHL synthases (YpeI and YspI) in Y. pestis and with the variety in structure. Secondly, NTL4 and BB170 were used to analyze the change of concentration of AHLs and AI-II during the whole growth phase of the Y. pestis wild strain and 6 kinds of QS-related mutant strains so as to confirm the envelop of function of two types of QS systems and simultaneously to reveal the internal regulatory mode between two regulatory systems in type-I QS system. Thirdly, the multicellular forming state and quantity in LB and TMH culture medium of wild strain and QS mutant strains were systematically analyzed, which led to the analysis of correlation between type-I QS system and multicellular phenomenon. Finally, the comparative proteomics analysis of type-I QS system was performed to reveal how type-I QS system influence the survival and virulence of Y. pestis under the condition of high cell density.Results:Firstly, the qualitative detection results agreed well with genetic prediction, that is, type-I and type-II QS coexisted in Y. pestis. The identification of HPLC-MS-MS showed that there were four kinds of AHLs synthesized by Y. pestis, namely C6-HSL, C8-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL. AHL synthase YpeI is in charge of synthesizing 3-oxo-C6-HSL, while YspI is in charge of synthesizing C6-HSL, C8-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL. Secondly, the semi-quantitative analysis of reporter bacteria showed that AHL did not expire during the period ranging from early logarithmic phase to late static phase. The peak emerged at 2-3 hours after entering into static phase and lasted for about 10 hours. While AI-II did not expire during the period ranging from early logarithmatic phase to static phase. The peak came out at some moment of middle-late logarithmic phase and disappeared before reaching the static phase. The choice of Y. pestis between type I and type II QS was thoroughly directed by its own growth state. According to the difference of AHLs production among wild strain and mutant strains, it could be predicted that there existed self-regulatory mechanism in type-I QS system, that is to say, there existed negative regulatory circuits in both ypeI/ypeR and yspI/yspR regulatory system and meanwhile ypeI/ypeR regulatory system lie on the upper reach and had a negative regulatory effect on yspI/yspR. Thirdly, the phenotypic analysis of the multicellular forming state and quantity of wild strain and mutant strains showed that QS was directly related to the multicellular forming and regulatory mechanism of ypeI/ypeR system played a decisive role. Finally, the comparative proteomics analysis indicated that type-I QS had an obvious positive regulatory effect on self-protection, energy transport and energy metabolism of Y. pestis, which enhanced the survival potential under the condition of high cell density in static phase and probably increase the virulence of Y. pestis indirectly.Conclusion:Through the systematic analysis of QS system in Y. pestis, it can be confirmed that both type-I and type-II QS system exist in the Y. pestis. They have different envelop of function and influence different phenotypes of Y. pestis through the synthesis of different kinds of autoinducer. And type-I QS system have an obvious positive regulatory role on self-protection, energy transport and energy metabolism of Y. pestis. Based on the above mechanism, the survival capability of Y. pestis under the condition of high cell density is enhanced and its virulence may be increased indirectly. However, no positive regulatory effect on known toxin has been found.
|
PDF Full Text Request