| Listeria monocytogenes(Lm)is an important foodborne zoonotic pathogen,which mainly enters the digestive tract of the host through contaminated food.With the help of several surface virulence factors,Lm could break through intestinal barrier,blood-fetal barrier and blood-brain barrier,leading to high mortality rate after infection.Wall teichoic acids(WTAs)are the important anionic glycopolymers on the surface of Lm cell wall,and covalently bound to the peptidoglycan layer with the long chain of phosphodiester-linked polyol units(ribitol-phosphate,RboP).According to the N-acetyl glucosamine(GlcNAc)glycosylation modification of RboP chain contains,WTAs can be divided into type Ⅰ WTAs and type Ⅱ WTAs.The specific glycosylations of WTAs is the main determinant of the antigenic characteristics and immunogenicity of Lm,contribute to Lm cell homeostasis,drug resistance,host immune response and virulence.The glycosylation modification of Lm type I WTAs contains rhamnose and GlcNAc.At present,the pathogenic mechanism mediated by WTAs GlcNAc glycosylation has been reported,but the biological characteristics and immunomolecular mechanism of Lm WTAs rhamnosylation have not been fully revealed.In this study,we constructed gene rmlT mutant strains of serotype 1/2a Lm EGD-e to indicate its effection on the antigenic and growth characteristics of Lm.At the same time,we extracted and purified WTAs from EGD-e and rmlT mutant strains,and identify their detailed structures under the analysis of ultraperformance liquid chromatography-coupled electrospray ionization tandem mass spectroscopy(UPLC-MS/MS).Subsequently,we used the intestinal epithelial cell Caco-2,macrophage RAW264.7,mouse primary bone marrow-derived macrophages(BMDMs)and C57BL/6N mice as infection models to determine the functions of Lm WTAs rhamnosylation in bacterial invasion,colonization and host inflammatory response.This study aims to reveal the pivotal role of WTAs rhamnosylation in the pathogenesis and immunity of Lm,and lay a theoretical foundation for the prevention and treatment of listeriosis.1.Construction and biological characteristics of wall teichoic acids rhamnotransferase gene rmlT mutant strainIn this study,we first explored the bioinformatics of rmlT gene and predicted its protein structure and function.rmlT encodes an extracellular protein RmlT,composed of 623 amino acids,and its three-dimensional structure is similar to Staphylococcus aureus WTAs glycosyltransferase TarS.The predicted results of interacting protein interaction showed that RmlT may interact with rhamnose synthase lmo1084,glycosyltransferase lmo1090,lmo1744 and lmo2550,as well as teichoate synthase lmo1081,lmo1082 and lmo1083,which suggested that RmlT is involved in the biosynthesis of Lm WTAs modified by rhamnose and GlcNAc.Furthermore,we constructed WTAs galactosylation deficient strain EGD-e ΔrmlT,which lost rhamnose glycosylation modification of WTAs,and its revertant strain EGD-eΔrmlT::pIMK2(rmlT)by homologous recombination technique provided biomaterials for analyzing their related functions.At the same time,the characteristics of bacterial antigen of Lm are determined by plate agglutination test,and the results showed that EGD-e ΔrmlT did not agglutinate with Lm OI antiserum antibody.In addition,the structure and glycosylation modification of Lm WTAs are analyzed and identified by UPLC-MS/MS.The results showed that the deletion of rmlT gene leaded to the loss of rhamnose glycosylation modification in WTAs,and proved that rmlT gene encode rhamnose transferase of Lm EGD-e WTAs.In order to study the effect of rhamnose glycosylation modification of WTAs on the growth and reproduction of Lm,we measured the growth curves of wild strain EGD-e,rmlT mutants EGD-e ΔrmlT and EGD-e ΔrmlT::pIMK2(rmlT)in BHI medium.The results manifested that there is no significant difference between wild strain and mutant in BHI medium,indicating that rhamnose glycosylation modification of WTAs make no impact on the growth of Lm.2.Characteristics of proinflammatory immune response of rhamnosylated-wall teichoic acidsIn this study,macrophage RAW264.7,BMDMs and C57BL/6N mice are used as infection models to explore the characteristics of WTAs rhamnosylation mediate Lm-induced host inflammatory response,organ colonization and virulence.We first measured the adhesion and invasion ability of Lm by intestinal epithelial cell Caco-2,macrophage RAW264.7 and BMDMs.The results showed that the invasion ability of rmlT deletion strain to Caco-2 cell line is significantly decreased,and the adhesion and invasion ability to BMDMs are both significantly decreased.Secondly,we explored the immunological characteristics of rhamnose glycosylation modification of Lm WTAs.The results showed that compared with the deleted strain,rhamnosylation of Lm wild-type WTAs could significantly upregulate the transcriptional expression of pro-inflammatory cytokines such as IL-6,IL-1β and TNF-α in macrophages RAW264.7 and BMDMs,and downregulate the transcriptional expression of anti-inflammatory cytokines IL-10.In addition,the infection of rhamnosylation WTAs and wild-type Lm on BMDMs could significantly upregulated the secretory expression level of IL-6 and TNF-α from the supernatant of BMDMs cells.Furthermore,in this study,we detected the activation level of NF-κB signal pathway.The results showed that rhamnose glycosylation modification of WTAs promoted the activation of NF-κB signal pathway.At the same time,we used Western blotting to detect the phosphorylation levels of p65 and IκBα,which are the key proteins of NF-κB signaling pathway.The results indicate that Lm WTAs rhamnosylation enhance the phosphorylation levels of p65 and IκBα.Furthermore,we measured changes of body weight and the transcription level of inflammatory cytokines after WTAs stimulation.The results showed that rhamnose glycosylation modification of WTAs could significantly upregulate the transcriptional expression of IL-6,IL-1β and TNF-α in vivo.Secondly,we determined the colonization ability,transcription level of inflammatory cytokines in organs and histopathological changes in mice,which are infected by EGD-e,EGD-e ΔrmlT and EGD-e ΔrmlT::pIMK2(rmlT)strains.The results showed that the colonization ability of EGD-e wild strain and rmIT revertant strain,which both containing rhamnose modified WTAs,in liver and spleen was significantly higher than that of EGD-e without WTAs rhamnosylation,suggested that the rhamnose glycosylation modification of WTAs can promote the colonization of Lm and contribute to the survival of Lm in vivo.Then we detected the transcription levels of inflammatory cytokines in liver and spleen of mice by quantitative real-time PCR.The results showed that WTAs rhamnosylation mediates Lm upregulate the transcription level of pro-inflammatory cytokines IL-6,IL-1β and TNF-α in liver and spleen of mice.Histopathological analysis showed that rhamnose glycosylation modification of WTAs increased the infiltration and necrosis of inflammatory cells in liver and spleen of mice,and promoted the occurrence of tissue inflammatory reaction.To sum up,the rhamnose glycosylation modification of WTAs is an important O-antigen determinant of serotype 1/2a Lm EGD-e strain,contributing to enhance Lm adhesion and invasion ability to host cells and the colonization in the host.This study preliminarily reveals the molecular mechanism of WTAs rhamnosylation promoting host immune response,indicating that rhamnose glycosylation modification of WTAs can activate NF-κB signaling pathway and upregulate the transcriptional expression and secretion of downstream pro-inflammatory cytokines.Therefore,WTAs rhamnosylation is of great significance for the prevention and treatment of listeriosis. |
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