The Function Of NTSN₁086 And NTSN₂723 Genes In Serovar 4b Listeria Monocytogenes

Listeria monocytogenes?Lm?is a Gram-positive foodborne pathogen which can cause infections in the elderly,pregnant women,infants,and immunodeficiency patients.According to the bacterial somatic and flagellar antiseras,Lm can be divided into 14 serovars among which strains of serovars 4b,1/2b and 1/2a cause most human clinical listeriosis.Listeria antigenicity is conferred by specific glycosyl-modified wall teichoic acids?WTAs?.As the important surface sugar polymers on the cell wall of Gram-positive bacteria,WTAs participate in the surface anchoring of virulence factors on the cell surface,are the important virulence factors of Lm.play the crucial role in the invasion and virulence to the host cell.Due to modifications by a variety of glycosidic substitutions,two main WTA structural types exist:Type ? WTAs,be decorated with terminal rhamnose or GlcNAc and Type ? WTAs,be decorated with glucose and/or galactose.The mechanism of disaccharide modified type ?WTAs in listeria infection and pathogenesis remains to be elucidated.In this study,based on the construction of NTSN₁086 and NTSN₂723 mutant and revertant strains,the somatic antigenic,morphological and structural characteristics and stress resistance of Listeria were studied.In addition,intestinal epithelial cells,cervical cancer cells and mice were used to evaluate the virulence.Functional analysis of WTA glycosylation is beneficial to reveal the molecular mechanism of Lm infection and pathogenesis,and provide new insights into the infection and prevention of Listeriosis.1.Construction of NTSN 1086 and NTSN₂723 mutant strainsBioinformatics analysis of NTSN₁086 and NTSN₂723 showed that these two genes encoded galactosyltransferase and glucosyltransferase.NTSN₁086 and NTSN₂723 were highly homologous to the Listeria sequence of serovar 4b in the NCBI database and highly conserved genes in serovar 4b strains.Knockout of homologous genes resulted in the deletion of galactose and glucose in WTAs.Structures and functions of NTSN₁086 and NTSN₂723 need to be further elucidated,and the mechanism of disaccharide modified WTAs in listeria infection and virulence remains to be elucidated.The WTA-galactosylation deficient strain NTSN?1086,its revertant strain NTSN?1086::1086,WTA-glucosylation deficient strain NTSN?2723 and double genes deletion strain NTSN?1086?2723 were successfully constructed,which provided biological m aterials for studying the role of WTA glycosylation.The somatic antigenic properties of bacteria were determined by slide agglutination test and laser confocal microscope.The results showed that NTSN?1086 and NTSN?1086?2723 could not agglutinate wi th listeria O antigen type 4,and their binding ability with fluorescent labeled antibod y weakened.However,both the NTSN?2723 and the wild strain could agglutinate wi th the antibody,suggested that galactosylated WTA played a key role in the Lm anti genic properties.2.The biological characteristics of NTSN 1086 and NTSN 2723In order to study the effect of WTAs glycosylation on the morphology,structure and growth of bacteria,the composition of WTAs was detected by Native PAGE,and the cell morphology was observed by scanning electron microscope.The results showed that the absence of NTSN 1086 led to incomplete cell division,but had no significant effect on the growth of bacteria in BHI medium.In addition,this study also investigated the ability of glycosylated WTA to form biofilm and the resistance to antimicrobial peptides.The deletion of NTSN 1086 significantly affected the biofilm forming ability,and the resistance to the antibacterial peptides CRAMP and LL37 decreased significantly.However,the absence of NTSN 2723 had no significant effect on the biolilm formation and the resistance to antimicrobial peptides.This suggests that galactosylated WTA plays an important role in the morphological structure and stress resistance of bacteria.The binding ability and localization of virulence factors on bacterial surface were also examined.The results showed that in the strains lacking WTA-Gal,the expression level of actin aggregation factor ActA,internalin protein InlA,autolysin protein Ami and internalin protein InlB were decreased in the surface extracts and strongly increased in the secreted extracts.In addition,it was found that NTSN?1086 had a decreased ability to aggregate actin,and could not form actin tails,so they could not migrate between cells,indicating that WTA glycosylation with Gal is essential for the surface association of the processed form of virulence factors.However,the absence of NTSN₂723 had no effect on the surface anchoring ability of virulent proteinsHela cell line and Caco-2 BBe cell line were used as in vitro infection models.The results showed that NTSN?1086 and NTSN?1086?2723 significantly reduced the ability of adhesion,invasion and replication to Hela cells than wild strains.The ability of NTSN?1086::1086 restored to a level similar to that of the wild strain.The adhesion of NTSN?2723 was not different from that of wild strains,but the invasion and replication ability was significantly reduced.The ability of NTSN?1086?2723 was significantly lower than NTSN?2723 and NTSN?1086.From the results of bacterial adhesion,invasion and replication of Caco-2 BBe cell lines,it was observed that the invasion ability and replication ability of NTSN?1086 were extremely significantly lower than wild strains,and the replication ability of NTSN?2723 was significantly reduced,while the replication ability of NTSN?1086?2723 was significantly lower than NTSN?1086 and NTSN?2723.Thus,galactose and glucose in wall teichoic acid play a common role in Lm invasion and replication,but galactose is more important than glucose.Using mice as an in vivo oral infection model,the colonization abilities of NTSN 1086 and NTSN 2723 was measured.In addition to stool,the colonization ability of NTSN?1086 in ileum,colon,mesenteric lymph nodes,liver and spleen was significantly decreased.The bacterial counts of NTSN?2723 were significantly lower in mesenteric lymph nodes and liver.The colonization ability of NTSN?1086?2723 was significantly lower than that of NTSN?1086 in colon and NTSN?2723 in liver,colon and stool.Therefore,NTSN₂723 and NTSN₁086 are important virulence-related factors of Lm,the former plays an important role in colon,mesenteric lymph nodes and liver,and the latter plays an important role in colonizing multiple organs of Lm.In summary,the biological characteristics of NTSN₁086 and NTSN₂723 mutant strains pointed to that NTSN₁086 played a key role in the somatic antigenic properties of Lm bacterium;both of them were involved in the maintenance of bacterial morphology and structure and stress resistance;the colonization effects of the two genes in different organs in vivo were different,and synergistically played an important role in the infection and virulence of Listeria.Therefore,NTSN 1086 and NTSN 2723 are important virulence-related factors of Lm,which are expected to become new antimicrobial drug targets and have important clinical and public health significance for the prevention and control of listeriosis.