Listeria Monocytogenes:Biological Functions Of Aminopeptidase Lmo1711 And Flagellar Structural Protein FlhB

Listeria monocytogenes?LM?is an intracellular pathogen of gram-positive bacteria and is widely found in the environment.L monocytogenes can pass through the gut barrier,spread through the bloodstream and reach the liver,spleen,central nervous system and fetus,causing sepsis,encephalitis,meningitis,gastroenteritis,and abortion in pregnant women.Listeriosis is one of the most serious human foodborne infections with the mortality rate as high as 30%.In the process of infection,LM needs to express and secrete a variety of virulence factors to complete infection invading the host cells.L.monocytogenes requires different secretion systems to secrete virulence factors to the extracellular It is known that the Sec secretion system and the Tat secretion system are mainly responsible for secretory functions,whether flagellar secretion system is also involved in the secretion of specific factors has not been reported.Therefore,this article mainly elaborates:1.The enzymatic characteristics of L.monocytogenes aminopeptidase Lmo1711 and its biological role in cell infection.2.The biological function of L.monocytogenes flagellar structural protein FlhB.1.Enzymatic characteristics of Listeria monocytogenes aminopeptidase Lmo1711 and its involvement in bacterial infection Our bioinformatics analysis predicted that Lmo1711 belonged to aminopeptidase type II,M29 family,which could hydrolyze amino acid N-terminal residues.Enzyme activity analysis showed that Lmo1711 had strong aminopeptidase activity,and the binding and catalytic ability of different substrates differed greatly.Among the p-nitroaniline substrates,the corresponding Kcat/Km for Leu-pNA,Arg-pNA,and Lys-p NA was 3.149×10⁵ min^-11 M^-1?1.536×10⁵ min^-11 M^-1and 1.035×10⁴ min^-11 M^-11 respectively,it could be seen that Lmo1711 had the highest catalytic efficiency for Leu-p NA,ie the highest affinity for leucine residues.Lmo1711 aminopeptidase activity was metal ion dependent,and plenty of metal ions,such as Co²⁺,Cd²⁺,Zn²⁺could significantly enhance its activity,among which Co²⁺activation effect was the most significant.Enzymological analysis showed that the conserved key amino acid active sites presented in Lmo1711?Glu250,Glu316,His345,Tyr352,His378,Asp380?determined the catalytic activity of Lmo1711.The biological function study found that the ability of bacterial growth of?lmo1711was not affected,whereas the ability to invade and proliferate in infected Caco-2 cells was significantly lower than that of the wild strain.In addition,the ability of?lmo1711 to migrate in L929 cells was significantly weakened.We here firstly demenstrate that Listeria monocytogenes Lmo1711 is a member of the M29 aminopeptidase family,which has a strong catalytic activity,and has different degrees of dependence on metal ions,what's more,to some extent it is involved in bacterial infection in cells.2.The biological function of Listeria monocytogenes flagellar structural protein FlhB Our study found that Listeria.monocytogenes flagellar structural FlhB?encoded by the lmo0679 gene?was involved in bacterial flagellar synthesis and motile,Listeria did not form flagella and lost motile ability once flhB was deleted,which resulted in a decrease in transcriptional levels of many flagellar related genes,such as motA,flaA,fliN,fliI,flgC and fliK.Knockout of flhB resulted in decreased expression of Fli M,FliY and FlaA.According to the relationship between bacterial flagella and type III secretion system,in this study,the flagellar structural element FlhB was the main subject,and the association between the flagellar structure and the protein secretion function was explored for the first time in L.monocytogenes.In this study,differential protein profiling of secreted proteins in?flhB and?fliS was performed using isobaric tags for relative and absolute quantification?iTRAQ?.The iTRAQ results showed that in the absence of flhB and fliS,the secretion of a large number of flagellar and non-flagellr proteins decreased.At 37°C,38 proteins was up-regulated and 39 proteins was down-regulated in?flhB,25 proteins was up-regulated and the same amount of proteins was down-regulated after in?fliS.At 30°C,29 proteins was up-regulated and 62 proteins was down-regulated in?flhB.23 proteins was up-regulated and 57 proteins was down-regulated in?fliS.It could be seen that at 30°C,the number of down-regulated proteins in?flh B and?fliS was much greater than that at 37°C.The classification of COG?Cluster of orthologous group?showed that these differential proteins were mainly involved in translation,ribosome structure and biogenesis,sugar transport and metabolism,etc.;GO?Gene ontology?analysis identified that the differential proteins were mainly cellular components?27.74%?,macromolecular complexes?12.19%?and organelles?12.01%?.After querying Uniprot et al.'s protein information website and related literature,we selected a few proteins reported rarely in L.monocytogenes,such as hook-regulatory protein FliK,flagellar filament protein FlaA and cell-division regulation protein DivIVA for western blot verification.The WB results showed that the secretion of DivIVA,FliK and Fla A decreased significantly in?flhB at 30°C,and the secretion of Fli K and FlaA decreased significantly in?fliS.Among them,DivIVA is related to the division and proliferation of bacteria.This study firstly reveals that L.monocytogenes FlhB is involved in bacterial migration,flagellar synthesis and the secretion of flagellar and non-flagellar proteins,providing a preliminary experimental basis for the further exploration of the non-classical protein secretion mechanism and pathogenesis of L.monocytogenes.In summary,we demonstrate for the first time that?1?Listeria monocytogenes aminopeptidase Lmo1711 belongs to the metal ion-dependent M29 aminopeptidase family members,and participates in bacterial infection and migration in cells due to bacterial virulence related.The research results are of great significance for the thorough improvement and exploration of new virulence factors and pathogenic mechanisms of Listeria.?2?The biological function of Listeria flagellar structural protein FlhB is explored,which lays a key foundation for the further study of biological functions of Gram-positive bacterial T3 SS and new secretory proteins.