The Mechanisms Of Two-component Systems Regulating Lysozyme Resistance And Virulence In Listeria Monocytogenes

Listeria monocytogenes is a Gram-positive foodborne pathogen that is able to contaminate a variety of food products.Ingesting contaminated food can lead to gastroenteritis,meningitis,sepsis and even death.As a high-efficiency natural preservative,lysozyme has been widely used in the food industry.In addition,as a cornerstone of innate immunity,lysozyme is essential for host defense.L.monocytogenes has evolved lysozyme resistance which is associated with pathogenicity.Whereas the genes of L.monocytogenes lysozyme resistance have been sporadically studied,much is still unknown about the mechanisms.The exploration of the lysozyme resistance mechanisms of L.monocytogenes could not only deepen our understanding of antimicrobial resistance in pathogenic bacteria but also provides solid rationale for the development of high-efficiency antimicrobials and clinical investigation of listeriosis.In this study,we leveraged transposon sequencing(Tn-seq)in the L.monocytogenes to identify genes that are involved in lysozyme resistance.Subsequently,we focused on two-component systems to identify their role in lysozyme resistance and pathogenicity of L.monocytogenes and to explore their mechanisms of action via transcriptome sequencing(RNA-Seq).We also revealed the role of these twocomponent systems in L.monocytogenes infection breaking through the protective barrier of gut microbiota.The main contents and results are as follows:(1)The high-density transposon library of L.monocytogenes was constructed and a genome-wide screen for essential genes was performed by Tn-seq.A mariner transposon library of L.monocytogenes was constructed by a thermo-sensitive plasmid.The study found that the mutation library contained 43,793 mutant strains,with an average of 67 bp in the genome having a transposon insertion.And 637 essential genes of L.monocytogenes were identified,whose functions clustered in translation,metabolism,and cell wall/envelope synthesis.(2)The lysozyme resistance-related genes of L.monocytogenes were identified via Tn-seq.We identified 44 genes and 2 non-coding RNAs involved in lysozyme resistance.In addition to several known genes,many novel lysozyme resistance-related genes have been characterized including four TCS genes.We used transcriptome analysis to identify genes regulated by the exposure to lysozyme,142 differentially expressed genes were screened.The analysis showed that L.monocytogenes exposure to lysozyme decreases cellular injury by reducing the net negative charge of the bacterial cell surface,decreasing secretion of autolysins,and inhibiting cell division.(3)Four lysozyme resistance-related TCS mutants(?deg U,?vir R,?ces R,and?yyc I)were generated by homologous recombination and Cre-lox system to investigate their properties.Results show that DegU,Vir R,Ces R,and Yyc I were involved in lysozyme resistance with different mechanisms,and contributed to cell wall homeostasis.DegU and Vir R affect the dual activity of lysozyme(muramidase and cationic antimicrobial peptide)against L.monocytogenes,Vir R and Yyc I affect the lysozyme resistance of L.monocytogenes by reducing the net negative charge on the cell surface.(4)We used different mice infection models to probe the relationship between virulence and lysozyme resistance and explored the possible virulence factors in the infection route of L.monocytogenes.(I)Oral infection of SPF mice was used as a natural infection route model.After ingesting contaminated food,L.monocytogenes entered the host intestine,crossed the intestinal barrier,and then spread to the liver and spleen.In this model,the lysozyme-sensitive TCS mutants exhibited attenuated virulence.(II)The SPF mice intraperitoneal infection model completely bypasses the intestinal phase of infection.In intraperitoneally infected mice,?deg U and ?vir R mutants were attenuated,the ?ces R have no significant changes in virulence,but ?yyc I have enhanced virulence,indicating that several factors attenuated the virulence of lysozyme-sensitive mutants in the stage of gastrointestinal infection.(III)Unlike the SPF mice oral infection model,the antibiotic-treated(Abx)mice oral infection model abolished the effects of the gut microbiota on L.monocytogenes during infection.The infection results of Abx mice model are consistent with the intraperitoneal infection of SPF mice,indicating that gut microbiota provides first line defense against L.monocytogenes infection.Enterococcus strains that only presented in SPF mice but not in Abx mice were identified against L.monocytogenes.Consistent with the result of Abx mice experiments,the TCS mutants showed increased sensitivity to these Enterococcus.The results of these animal model studies suggest that two-component systems related to lysozyme resistance of L.monocytogenes regulate virulence through the gut microbiota.(5)The mechanisms of TCSs regulating lysozyme resistance and virulence were investigated by transcriptome sequencing.The results showed that DegU,Vir R,Ces R,and Yyc I mediate lysozyme resistance and virulence of L.monocytogenes by regulating the expression of different lysozyme resistance-and virulence-associated genes.DegU mediates L.monocytogenes virulence and lysozyme resistance by regulating the expression of pgd A,oat A,pbp4,and pbp X.The down-regulated expression of dlt ABCD,mpr F,and anr AB genes may lead to reduced virulence and enhanced lysozyme sensitivity of ?vir R mutants.Ces R regulates down-regulation of the virulence-related genes orf2420,ces K,lmo2459,and lmo2522.Yyc I regulates several virulence-related genes,such as spo VG,inl A,inl B,inl H,act A,etc.Moreover,DegU,Vir R,Ces R,and Yyc I could all regulate the genes related to flagellar synthesis,and the biofilm formation ability of the four lysozyme-sensitive mutants was decreased.