Role Of Salmonella Enteritidis Gene Rfbg,rfbH And AvrA And Their Molecular Mechanisms

Salmonellosis is the important zoonosis.It is the general term of human and animal diseases caused by Salmonella infection.It is usually characterized by nausea,vomiting,abdominal pain,diarrhea,fever and headache in humans.Remarkably,numerous lines of evidence have indicated that Salmonella infection is also involved in extra-intestinal infections,like urinary tract infections,septicemia,systemic lupus erythematosus,rheumatoid arthritis and endocarditis.Salmonella enterica serovar Enteritidis(Salmonella Enteritidis,S.Enteritidis,SE),which has extensive hosts,is one of the most common serotypes of Salmonella infection in humans despite the implementation of control and prevention measures.Infection caused by Salmonella Enteritidis results in significant economic loss to the poultry industry and places a substantial burden on the healthcare system.Although Salmonella Enteritidis is an important pathogen with a public health concern,the majority of studies still focus on Salmonella Typhimurium.It neesd to enhance the basis studies on the pathogenesis,prevention and control of Salmonella Enteritidis.Salmonela possesses protective outer membrane,with its external leaflet composed of lipopolysaccharides(LPS).Recently studies show that LPS is responsible for smoothness,virulence,flagellar assembly and for mounting cross reactivity.LPS has three primary components.A core polysaccharide unit,which ligated the O-antigen component and lipid A component.The completeness of the O-chain antigen determines whether the LPS is smooth or rough.The mutations of LPS biosynthesis related genes may lead to bacteria possessing a rough LPS.However,related genes and their mechanisms still need further study.Salmonella possesses a range of effector proteins that are translocated into the host cells via a type ? secretion system(T3SS).These effector proteins are generally assumed to influence the host's cellular functions to facilitate Salmonella invasion and survival.AvrA is one of the Salmonella effector,which owns acetyltransferase activity and inhibits the host c-Jun N-terminal kinase(JNK)/AP-1 and NF-?B signaling pathways.Through these approaches,AvrA inhibits the host inflammatory response and stabilizes the intestinal tight junctions to the benefit of Salmonella survival,but its molecular mechanism still remains to further explore.To analyze the pathogens through the molecular level is a conventional and promising method for understanding the pathogenesis mechanism of bacteria.In this study,we used monoclonal antibody against somatic O:9 antigen(O:9 mAb)of Salmonella to screen STM(signature-tagged mutagenesis)bank of S.Enteritidis mutants libraries to identify novel factors associated with rough variation,and it was found that the genes rfbH and rfbG were involved in this process.Furthermore,we determined the biological functions of these genes and their"DIVA(differentiation of infected and vaccinated animals)" capacity in S.Enteritidis.Moreover,by using SE-WT,SE avrA mutant and SE avrA complementary strain to infect cell cultures,organoids and mice model,the physiologically related effects and molecular mechanism of AvrA regulation of autophagy in intestinal epithelial cells were further explored.1.Screening and identification of novel determinants involved in smooth-to-rough transition of Salmonella enterica serovar EnteritidisIn this study,we used homemade O:9 mAb and acriflavine to screen S.Enteritidis signature-tagged transposon mutants to identify novel factors involved in smooth-to-rough transition.And we found O:9 mAb has no reaction with three mutant strains in the STM library.Acriflavine performed agglutination assay was used to confirm whether these mutants were rough strains,.The results showed an obvious agglutination reaction between two mutants and acriflavine.By a PCR-based method for specific amplification of transposon-flanking sequences,the inactivated genes were identified as rfbG and rfbH.Both genes are from the rfb gene cluster of S.Enteritidis and are responsible for biosynthesis of LPS.The third mutant,which has weak agglutination reaction with acriflavine,was identified to be rfc mutant.The rfc gene,which is independent of the rfa and rfb gene clusters,encodes an O-antigen polymerase.Furthermore,we determined the biological functions of rfbG and rfbH gene in S.Enteritidis.(1)rfbG mutant and rfbH mutant showed similar growth cuve and biochemical characteristics compared with SE-WT.(2)The results of agglutination assay,LPS staining and auto-aggregation test showed that rfbG mutant and rfbH mutant have typical rough characteristics,like affecting agglutination reaction with O:9 mAb or acriflavine,having auto-aggregation phenomenon and resulting in deep loss of LPS synthesis in S.Enteritidis.(3)Results obtained from motility assays indicate that the deletion of rfbG or rfbH gene resulted in variation in swimming and swarmming motility.(4)The stress resistance data showed that disruption of the rfbG or rfbH gene increased susceptibility of the S.Enteritidis strain to oxidative,alkali and didecyl dimethyl ammonium bromide and iodine complex solution.And high sensitivity to alcohol and heat shock were demonstrated in wild type,rfbG mutant and rfbH mutant strain.These results suggested that not only rfbH and rfbG gene,but also rfb gene cluster are essential for LPS biosynthesis and flagellar assembly in S.Enteritidis.And it provided the biologic materials for studying "DIVA" capacity of rfbH and rfbG mutant strains.2.Preliminary evaluation the "DIVA",safety and protective capacity of S.Enteritidis rough mutantsThe concept of DIVA vaccines based on the absence of some immunogenic antigens in the vaccine,but which are present in the wild type strain,has already been proposed for veterinary use.In this study,we determined the "DIVA",safety and protective capacity of rfbG mutant and rfbH mutant.Sera tests showed that,chicken sera collected from rfbG mutant strain group,rfbH mutant strain group and control group were considered Salmonella O:9 antibodies negative by both agglutination assay and ELISA test.In contrast,blood collected from the SE-WT group and spiC mutant group showed obvious reaction by agglutination assay,meanwhile were considered seropositive for Salmonella O:9 antibodies by ELISA test.Moreover,mouse sera collected from rfbG mutant immunization group also can not agglutinate with SE-WT culture from LB agar medium.Taken together,rfbG mutant and rfbH mutant can't elicit specific antibodies against O-antigens in vaccinated animals.Samples from those animals can not be detected by serologically diagnostic procedures,which were designed based on O-antigens of Salmonella.These results suggested that rfbG and rfbH gene loci may be used as vaccine marker to differentiate vaccination group from natural infection group in further study.Animal experiment results showed that rfbG mutant strain,which is safe for mammals and environment,can provide superior immune protection for immunized mice.Furthermore,C50041 ?spiC-rfbG::Tn5Km2(Cm)strain can inhibit the colonization of SE-WT in the liver of SPF chicken.It indicates that,rfbG mutant as a candidate vaccine is promising,and needs further study.In conclusion,we found rjbG and rfbH gene loci are potential DIVA vaccine marker.Meanwhile the STM combined with somatic O-antigen antibody-based screening technique demonstrated to be a powerful tool to explore LPS biosynthesis and to uncover novel distinguishable markers for the development of vaccine.3.Salmonella Enteritidis effector AvrA suppresses autophagy by targeting Beclin-1 proteinIn this study,we used SE-WT,SE avrA mutant and SE avrA complementary strain to infect HCT116 cell,organoids and C57BL/6 mice,in order to explore the physiologically related effects and molecular mechanism of AvrA regulation of autophagy in intestinal epithelial cells.In human intestinal epithelial HCT116 cells,the conversion of LC3 ? into LC3 ? was increased after SE avrA mutant infection compared to that after SE-WT or SE avrA complementary strain infection.And,p62 was decreased in the cells infected with the SE avrA mutant strain compared with the expression in cells infected with the SE-WT or SE avrA complementary strain.Meanwhile,HCT116 cells pre-treated with LysoTracker showed more lysosomes in the cells infected with the SE avrA mutant strain than in the cells infected with the AvrA expressed strains.Taken together,these data suggest that the Salmonella Enteritidis effector AvrA inhibits autophagy in vitro.Beclin-1 is a key molecular regulator of autophagy.Beclin-1 expression was decreased after AvrA expressed strain colonization in HCT116 cell.This difference was confirmed in both an organoid and mouse model.After treatment with the JNK inhibitor,the Beclin-1 expression did not differ between infection with and without AvrA in HCT116 cell.The results demonstrated that the S.Enteritidis effector AvrA inhibits the JNK/c-Jun/AP-1 signaling pathway to decrease Beclin-1 expression.And our data using AvrA WT and AvrA C186A mutant plasmids indicated that cysteine 186 is the key amino acid required for the AvrA regulation of JNK pathway and Beclin-1 expression.Furthermore,immunoprecipitation data showed that AvrA interacted with Beclin-1.Additionally,the AvrA C186A mutant plasmid showed reduced deubiquitinase activity,as evidenced by the removal of fewer ubiquitin moieties from ub-Beclin-1 compared that with the wild-type AvrA plasmid.Paneth cells are specialized epithelial cells that are primarily located in the small intestine.Our data showed that the normal expression pattern of the Paneth cells decreased in the mice infected with the AvrA present strain compared with that in the mice infected with the SE avrA mutant strain.The ileum,after infection with the AvrA present strain,contained an increased proportion of Paneth cells with disorganized or diminished granules or exhibited diffuse cytoplasmic lysozyme staining.Taken together,our data reveal a new role for AvrA in S.Enteritidis in the reduction of Beclin-1 protein expression through the JNK pathway and the attenuation of the autophagic response,for the benefit of Salmonella survival in host intestinal epithelial cells.Moreover,AvrA affects the function of Paneth cell granules,potentially by inhibiting autophagy.Our findings indicate an important role of S.Enteritidis effector in reducing host protein as a strategy to suppress autophagy and provide new insights into the pathogenic mechanisms of bacterial infection.