Two-Component-System Histidine Kinases Involved In Virulence Of Listeria Monocytogenes

Listeria monocytogens?Lm?is Gram-positive bacterium.It is an important food-borne pathogen designated by WHO and causes zoonosis-Listeriosis.Adaptability of Lm is strong.Lm is a saprophytic bacterium in the natural environment,while it becomes a pathogen when it invades into organism.Two-component signal transduction systems are important regulatory systems for bacterial adapting to the environment.TCS consists of histidine kinase?HK?and response regulator?RR?.HK senses external signals.RR regulates intracellular gene expression.This change of Lm from saprophytic bacterium to pathogen is closely related to some RRs in TCS of Lm.But reports of HK correlating with virulence are rare.Therefore,this study explored the correlation between HKs and the virulence of Lm so as to further understand the pathogenicity of Lm and provide theoretical basis for the prevention and treatment of listeriosis.The whole genome of Lm strain LM201 which isolated from food was sequenced and analyzed.There are 16 TCSs,14 pairs of HK/RR and 2 orphan RRs,in LM201.In this study,LM201 was used as the parental strain and 13 HK deletion mutant strains were constructed successfully.Growth curve,cell experiments and mouse virulence assay of the 13 HK deletion mutant strains were performed.This study provided a theoretical basis for further understanding the pathogenicity of Lm and the prevention and treatment of listeriosis.The specific experimental results are as follows.1.Construction of HK deletion mutant strainsUsing temperature-sensitive suicide plasmid pHT304-ts as vector and homologous recombination method,13 HK deletion mutant strains were successfully constructed,including S0106??cheA?,S0132??cesK?,S0142??phoR?,S0152??hssS?,S0162??kdpD?,S0172??agrC?,S0182??liaS?,S0192??lisK?,S01A2??yclK?,S01B2??resE?,S01C2??eutW?,S01D2??virS?and S01E2??yesM?.2.Growth curve of deletion mutant strainsThe growth curves of 13 deletion mutant strains at 37?were determined.The results showed that strains S0106??cheA?,S0142??phoR?,S0162??kdpD?and S0172??agrC?had lower bacterial load than LM201 at stable periods.The growth trend of other deletion strains was consistent with LM201.The growth curves of 13 deletion mutant strains at 5?were determined.The growth rate of strain S0192??lisK?was slower than LM201.The growth trend of other deletion strains was consistent with LM201.3.Cell test results of deletion mutant strainsIn the study of the effects of 13 deletion mutant strains on Caco-2 cells,results showed that the adhesion and invasion ability of S0192??lisK?,S01D2??virS?and S01E2??yesM?were significantly decreased.The adhesion ability of S0106??cheA?and S01A2??yclK?were significantly increased.The invasion ability of S01B2??resE?and S01C2??eutW?were significantly increased compared with the parent strain LM201.HBMEC cell experiment results showed that,the adhesion and invasion ability of S0182??liaS?were significantly increased compared with LM201.The adhesion ability of S0132??cesK?and S01B2??resE?were significantly decreased.And the invasion ability of S0172??agrC?was significantly increased.The results of RAW264.7 cell experiment showed that,the number of bacteria in cells of strains S01D2??virS?,S01B2??resE?and S01C2??eutW?decreased significantly compared with the parent strain LM201.It is suggested that the deletion of virS,resE or eutW gene would lead to the decrease of Lm viability in RAW264.7 cells.4.Mice virulence assays of deletion mutant strainsThe results showed that S0172??agrC?significantly decreased the bacterial load in liver at 24h,increased the bacterial load in liver and spleen at 48h,and decreased the bacterial load in liver and spleen at 72h.The bacterial load in liver and spleen of S0192??lisK?decreased significantly at 24h and 48h.S01E2??yesM?significantly decreased bacterial load in liver and spleen at 48h and spleen at 72h.The LD₅₀0 of 13 deletion mutant strains showed that,the LD₅₀0 of strains S0152??hssS?,S0172??agrC?and S01D2??virS?increased compared with their parents LM201?2.08×10⁵?,9.84×10⁵,10.37×10⁵ and 9.78×10⁵,respectively.Their virulence decreased by4.73,4.99 and 4.70 times respectively compared with LM201.The LD₅₀0 of strains S0142??phoR?,S0162??kdpD?and S0182??liaS?are 8.4×104,1.18×10⁵and 1.24×10⁵,respectively.The virulence increases compared with LM201?2.08×10⁵?.The results showed that the deletion of HssS,AgrC,LisK,VirS or YesM resulted in the decrease of Lm virulence.The deletion of LiaS resulted in the increase of Lm virulence.