Identification And Mechanism Of Virulence Genes In Listeria Monocytogenes

Listeria monocytogenes(L.monocytogenes,LM)is a food-borne pathogen with high lethality,posing a great threat to food safety.Previous studies have revealed many virulence factors and their mechanisms in LM.In order to deeply explore the pathogenic mechanism of LM and find new virulence factors,Tn-seq was used to identify virulence-related genes in genome-wide.Combined with transposon insertion and high-throughput sequencing,a high-density mutant library was constructed,containing at least 63,666 unique insertion mutants,and with average 46 bp per insertion.742 essential genes of LM involved in the basic life process of bacteria were identified by the ARTIST method.Many reported genes related to Caco-2 infection and some new virulence genes were identified by Tn-seq,including two phosphate transport gene lmo2248 and lmo2249.TheΔlmo2248 and Δlmo2249 mutants were found to have reduced viability in acid environments and in Caco-2 infection.In other conditions,including high bile salts,strong oxidation,tolerance in macrophages and serum,and biofilm formation were not weakened,but significantly enhanced.Consistent with the in vitro results,both the intraperitoneal injected mouse model and the food-fed infected mouse model showed enhanced virulence of the mutant strain.At the same time,cell morphology was significantly smaller in size than wild type were observed in the mutant strain.Transcriptomic studies and metabolite assays reveal molecular mechanisms underlying changes in virulence and morphology of the mutants.Lmo2248 and Lmo2249 are responsible for phosphate transport in a high-concentration phosphate environment.Gene deletion mutations lead to the up-regulation of Pst system and the decrease of intracellular phosphate concentration;PTS system were down-regulated,the ability to uptake and utilize glucose was reduced;pyruvate synthesis was down-regulated,and the intracellular pyruvate content was reduced;and the virulence genes prf A,inl A and inl H were up-regulated,which affect the virulence of mutant strains.Cell shortening was associated with up-regulation of fts Z,and enhanced resistance to oxidative stress was associated with potassium and manganese transportrelated genes.In this paper,a high-density mutant library and Tn-seq data analysis process of LM were constructed,and new virulence factors lmo2248 and lmo2249 were identified,the relationship between the phosphate transport system and virulence were revealed.A new target for restricting the growth of LM and a foundation for functional genomics research of LM in furture were provided.