Preliminary Investigation Of CitB Family Two-component System In Avian-Specific Salmonella

Avian-specific Salmonella,namely Salmonella enterica serovar Gallinarum biovar Gallinarum(S.Gallinarum)and Salmonella enterica serovar Gallinarum biovar Pullorum(S.Pullorum),are important pathogens that can lead to seriously significant loss in the global poultry industry.These pathogens,as the causative agents for fowl typhoid and pullorum disease,continue to circulate and causing outbreaks in our country and many other countries over the world.They were also included in the national eradication bacterial diseases in the "National Medium and Long-term Animal Disease Prevention and Control Plan" in 2012.However,the pathogenic mechanism by which S.Gallinarum and S.Pullorum colonize the host and persistence in the targeted organs is remain still unclear.Two-component system(TCS)is the major type of signal transduction in many bacteria.Bacteria could apply the TCS to sense the environmental changes,regulate the downstream gene expression of virulence factors to maintain its survival in the context of environmental and host-associated interactions.They are named as the bacterial "IQ" and play an important role in bacterial growth and development,physiological metabolism,and pathogenicity.This study starts with the virulence-related Cit B family TCS of S.Gallinarum and S.Pullorum,including cit A-cit B(cit),dpi B-dpi A(dpi),dcu Sdcu R(dcu)three sets of TCS,through in vitro and in vivo investigations,systematically evaluate their biological function,compare the functional differences between different bacteria and different systems,and in further uncover the molecular mechanism for regulation of pathogenicity.The main research contents are as follows:1 Functional study of three sets of TCS in two avian-specific SalmonellaThe CRISPR-Cas9 system was used to construct gene-deleted strains of the cit,dpi,and dcu TCS,and with a comprehensive purpose,investigate for bacterial growth,usage of metabolites,biofilm formation,antimicrobial susceptibility,stress response,and cellular assays were performed.The results of the study showed that the cit,dpi and dcu TCS did not affect the growth of avian-specific Salmonella,metabolites usage,and the sensitivity of antimicrobial susceptibility.Under aerobic conditions at 42°C,the biofilm-forming ability of the cit and dpi mutants of S.Gallinarum was decreased in compared with the wild type strain;while under aerobic conditions at 20°C,the biofilmforming ability of the cit and dpi mutants of S.Pullorum was increased in compared with the wild type strain of S.Pullorum.In the stress assays,the survival ability of dpi and dcu mutants of S.Gallinarum increased under acid stress and oxidative stress,while the dcu mutant of S.Pullorum showed a lower level of susceptibility under oxidative stress.In the cellular infection experiment,the survival ability of HD-11 cells decreased for 20 hours in dcu mutant of S.Gallinarum.2 Investigation on the pathogenic role of TCS in avian-specific SalmonellaBased on the construction of gene-deleted strains,the pathogenicity analysis of avian-specific Salmonella wild strains with mutants was carried out to evaluate the role of the pathogenic mechanism in Cit B family.Firstly,the chicken embryo infection model and chicken infection model were constructed respectively,then the differences in pathogenicity and colonization ability of Cit B family mutants were systematically analyzed.The results showed that the cit mutant of S.Gallinarum had enhanced liver colonization ability in the chicken embryo infection model,while in the chicken infection test of S.Pullorum,the dcu mutant strain had enhanced virulence.The dpi mutant showed different colonization in different tissues.In summary,this study demonstrated that the cit,dpi and dcu TCS of Cit B family participate and play a role in the pathogenic process of avian-specific Salmonella.Deletion of cit and dcu TCS can enhance Salmonella virulence,maybe it caused by the up regulation of other Salmonella virulence factors after the deletion,such as biofilm,fimbriae etc..The specific mechanism remains to be further research.The cit TCS affects the liver colonization ability of S.Gallinarum,which may be related to the ability to resist oxidative stress and heat stress in in vitro experiments.The dcu TCS affects the pathogenicity of S.Pullorum,but it is inconsistent with the trend in vitro,suggesting that the dcu TCS may interact with other pathways to affect the pathogenicity.The dpi TCS may be involved in signal transduction system mediated by oxidative stress,leading to different colonization.The inconsistency of the results of TCS cit B family suggests that the interaction mechanism between the bacteria and the host and the global regulatory system are complex,and a gene cannot be simply classified as a virulence positive regulatory factor or a negative regulatory factor.The results of one test cannot be extended to the entire pathogenic process.The above results in further clarify the role of the Cit B family in the pathogenic process by avian-specific Salmonella,provide new information for exploring the regulatory mechanism of Cit B family,and expand the understanding of the functional diversity,including pathogenic role,for TCS in Cit B family.