Screening And Functional Analysis Of Novel Virulent Genes In A Hypervirulent Listeria Monocytogenes

Lm and Listeria ivanovii?Li?are the only two species of Listeria that cause diseases,among which Li mainly infects ruminants,such as sheep and cattle,and rarely causes human disease,while Lm can infect both human and animals,causing invasive and gastrointestinal listeriosis.Lm was an intracellular parasitic pathogen,which can proliferate in multiple host cells?phagocytes,epithelial cells and endothelial cells etc.?and translocate to adjacent cells.Stringkly,it has the ability to cross gastrointestinal tract,placenta and blood-brain barrier,listeriosis-asscociated mortality is 20-30%.Nowdays,more than 70 virulent factors of Lm have been revealed the role in adhesion,invasion and intracellular proliferation,however,the molecular mechanism of infection and pathogenesis of Lm has not been fully elucidated and need to be further probed through high-throughput omics techniques.In this study,the function of newly discovered gene smcL in Lm XYSN was studied,the relationship between the expression product and the LLO hemolytic protein encoded by the hly gene was preliminarily discussed.Additionally,new virulent factors related to the virulence of Lm XYSN were screened with transposon mutagenisis technology via oral infection mice models.The discovery and functional analysis of the virulence related genes in Lm XYSN are helpful to reveal the pathogenic mechanism of high virulence of the strain and provide foundation and theory for the prevention and control of Listeriosis.1.Functional study of smcL gene in naturally recombinant Listeria monocytogenesBy comparing the gene sequence of smcL with the database,it was found that 97%of the gene homologous to smcL originated from Li,while Lm in the database haven't this smcL gene.This is the first time that the smcL gene has been harboured in Lm,and the smcL gene of Lm XYSN is possibly to be derived from Li.The sphingomyelin C?SmcL?encoded by smcL can specifically act on sphingomyelin and has hemolytic activity.Therefore,the expression of SmcL protein is likely to play an important role in enhancing the virulence of Lm XYSN.In order to investigate the function of Li-derived smcL,the four mutant strains?Lm XYSN?hly,Lm XYSN ?smcL and Lm XYSN ?hly?smcL?and two recombinant strains?Lm EGDe::pIMK2-smcL and Lm NTSN::pIMK2-smcL?were successfully constructed.CAMP-like haemolysis with R.equi tests showed that spatula-shaped hemolysis with R.equi was formed for strains harboring smcL gene.Additionally,it was found that the deletion of smcL gene resulted in 2-folds decreasion in hemolytic ability of Lm XYSN,interestingly,the hemolytic ability of recombinant strain Lm EGDe::pIMK2-smcL increased 4-folds,these results suggested that the expression of smcL enhanced the hemolytic ability of Lm XYSN.Infection experiment in vitro verified that the presence of smcL gene in Lm significantly increased the adhesion,invasion and proliferation of Lm to the non-phagocytic cell line Caco-2 BBE.The function of smcL in Lm XYSN was further evaluated in oral infection animal model.It is interesting to note that hly-smcL double deletion strain lost the invasion and colonization ability compared with hly deletion in mice after infection for 72 hours?P<0.01,P<0.05,P<0.05?.Importantly,the expression of smcL in NTSN significantly increased the invasion and colonization ability in ileum and further migrated to lymphoglandulae mesentericae.Together,these results demonstrate that the smcL as a unique gene in Lm XYSN plays a critical role in XYSN survival and favoring organs colonization,thereby smcL is one of the crucial factor on the enhancement virulence of XYSN.2.Functional study of the new virulent gene LMxysn₀620 of Listeria based on transpon mutation library technologyUsing BALB/C mice as animal model,the transposon mutation library of Lm XYSN was screened.From the mutant library,we randomly selected 500 mutant strains for oral infection test,and obtained 13 mutant strains with virulence decreasion more than 100 times.Through reverse PCR and gene sequencing,it was found that the transposon insertion sites of 13 mutant strains were mostly related to cell growth and metabolism?xysn₁358,xysn₁979,xysn₂165,xysn₂795,xysn₁812,xysn₂865,xysn₂030?,and some genes related to DNA transcription?xysn₀972?and protein modification?xysn₁491,xysn-1284?,and the function of two genes is unknown?xysn₂490,xysn₀620?.The virulence of XYSN₀620::Tn?LD50 = 107.85 CFU?was 588 times lower than that of Lm XYSN?LD50 = 105.08 CFU?,which indicated that xysn₀620 was involved in the process of Lm XYSN infection in mice.The deletion mutant strain Lm XYSN ?0620 was constructed,and the growth capacity of Lm XYSN ?0620 under different culture conditions was measured,results showed that there was no difference in the growth rate of the mutant strain and the parent strain in the BHI medium or the BHI medium with 1%Triton.The biofilm formation capacity of the mutant and parent strains was measured at different times and temperatures,results showed that the ability of Lm XYSN ?0620 to form biofilm at 42? decreased significantly at 48 h and 72 h?P<0.05,P<0.01?.Drug sensitivity test showed that the deletion of xysn₀620 gene affected the resistance to clindamycin,changing from susceptibility to intermediate.These results suggested that xysn₀620 possibly encoded components related with bacterial structure.Additionally,we found that the loss of xysn₀620 gene could cause a significant reduction in the ability of Lm XYSN to adhesion,invasion and proliferation of Caco-2 BBE cells by cell infection experiment in vitro?P<0.01,P<0.05,P<0.01?.Finally,the infection dynamics of Lm XYSN ?0620 in vivo were measured.It was found that the colonization time of Lm XYSN ?0620 in spleen was later that wild type strain,and the colonization ability in sleen and liver was significantly reduction than parental strain(Spleen:P<0.01,P<0.001;Liver:?P<0.05,P<0.001?.In a word,it suggested that XYSN₀620 plays crucial role in the anti-stress response,and participates the pathogenesis of invasion,colonization and infection of the host.