Molecular Mechanism Of The Effect Of Glycosyltransferase GalE On The Anchoring Of Internalin B In Listeria Mono-cytogenes

Listeria monocytogenes（LM）,is a gram-positive bacterium belonging to the phylum Firmicutes,Listeria.LM,as an extremely important zoonotic foodborne pathogen,can cause listeriosis in humans and a variety of animals.The successful invasion of LM into host cells is mainly mediated by virulence factors encoded by the first virulence island,including internalin A（InlA）,internalin B（InlB）,actin aggregation factor（ActA）,hemo-lysin O（Listeriolysin O）,metalloproteinase（Mpl）,and two lipases（PlcA and PlcB）.All of these major virulence factors need to be transported outside the bacterial cell through the secretory system and anchored to the bacterial surface or present outside the bacterial body in free form.Previous studies have shown that abnormal InlB anchorage signifi-cantly reduces the infectivity and pathogenicity of LM.Literature reports show that InlB is anchored to afichoic acid of the LM cell wall in a non-covalent bond form.In 4b sero-type strains,loss of galactosylation modification of pariefifificic acids（WTAs）signifi-cantly reduces anchorage quantification of InlB.Galactosylation of WTAs may be done by glycosyltransferases such as GalE,GalU,GttA,GtcA,and GttB,however only genes encoding GttA and GttB are specific in 4b serotype strains,while genes encoding GalE,GalU,and GtcA are persistently present in various serotypes LM strains.The effect of GalE on LM cell wall WTAs modification as well as InlB anchoring is unclear.Therefore,this study aims to explore the role and molecular mechanism of LM glycosyltransferase GalE in the process of InlB anchoring,and provide clues for further exploration of the pathogenic mechanism of LM.1.GalE mediates the pathogenicity of LM ScottA strainFirstly,the deletion strain of 4b serotype galE gene was constructed by homologous recombinant technology,and the galE gene complement strain was constructed by ex-pressing plasmid.Then,the minimum bacteriostatic test was used to assess whether the deletion of galE gene affected the sensitivity of LM to antibiotics;The anchorage of major virulence factors such as InlB on the surface of parental strains and galE gene deletion strains was analyzed by Western blot;The effect of galE gene deletion on LM infectivity and pathogenicity was evaluated by cell adhesion invasion assay and mouse virulence test.First,the 4b serotype strain ScottA was used as the parent strain to successfully con-struct galE gene deletion strains（ScottA-ΔgalE）and back complement strains（CΔgalE）.Growth assays showed that deletion of galE gene did not affect the growth capacity of LM in BHI medium.The minimum inhibitory concentration test showed that deletion of the galE gene did not affect the sensitivity of LM to penicillin and ampicillin.Adhesion invasion tests showed that the deletion of galE gene led to the adhesion and invasion ability of ScottA-ΔgalE strain to intestinal epithelial cells Caco-2 significantly lower than that of parent strain ScottA,and the adhesion and invasion ability of CΔgalE to Caco-2by the retrocomplement strain was comparable to that of parent strain ScottA.The results of Western blot showed that the surface-anchored InlB of the galE gene deletion strain ScottA-ΔgalE was significantly lower than that of the parent strain ScottA,and the InlB content in the culture supernatant was significantly higher than that of the parent strain,and there was no significant difference in the InlB distribution between the retrofit strain CΔgalE and the parent strain ScottA.The virulence test of mice showed that 48 hours after intraperitoneal injection infection,the bacterial load of the gene deletion strain ScottA-ΔgalE in the liver,spleen and brain of mice was significantly lower than that of the parent strain ScottA,and there was no significant difference in the bacterial load of the parent strain and the replenishment strain in the organs of infected mice.The survival rate of mice in the ScottA-ΔgalE infected group was significantly higher than that in the parent and supplement infected groups.The results of pathological section analysis showed that the degree of inflammatory cell infiltration in liver,spleen and brain tissues of mice infected with the gene deletion strain ScottA-ΔgalE was significantly lower than that in the parent and supplement strain infection group.The possible mechanism of GalE-mediated InlB anchorage and LM virulence was further elucidated by analyzing the effects of GalE on InlB anchoring and LM virulence in 1/2a,1/2b,1/2c and 4a serotype strains.2.Effect of glycosyltransferase GalE on LM internalin B anchoringFurthermore,the 1/2a serotype strain EGDe,the 1/2b serotype strain Hum Lm6,the1/2c serotype strain Hum Lm4 and the 4a serotype strain 850658-prfA_M7 were used as parental strains to construct galE gene deletion strains.The results of the minimum in-hibitory concentration test showed that the deletion of galE gene did not affect the sensi-tivity of other serotype strains to penicillin and ampicillin except for the 1/2a serotype EGDe strain.The results of the Western blot test showed that deletion of galE gene led to a significant decrease in the anchorage quantification of InlB on the surface of the 1/2a serotype strain EGDe and the 4a serotype strain 850658-prfA_M7,but significantly upreg-ulated the anchorage quantification of InlB on the surface of the 1/2c serotype strain Hum Lm4.The virulence test results showed that deletion of galE gene could significantly reduce the bacterial load of 1/2a serotype strain EGDe and 1/2c serotype strain Hum Lm4in the spleen of infected mice,but did not affect its bacterial load in the liver of infected mice.In summary,galE gene deletion and complement strains were constructed in this study,and it was shown through growth assays and minimum inhibitory concentration tests that galE gene deletion did not affect the growth capacity of LM and sensitivity to penicillin and ampicillin targeting the cell wall.However,the deletion of galE gene sig-nificantly reduced the anchoring ability of InlB on the surface of the 4b serotype strain,and significantly weakened the adhesion and invasion ability of LM to intestinal epithelial cells and the pathogenicity of mice.In the 1/2a serotype EGDe,the 1/2c serotype strain Hum Lm4 and the 4a serotype strain 850658-prfA_M7,the anchorage and pathogenicity of the LM surface InlB by galE gene deletion were inconsistent with those of the 4b serotype.This suggests that the effect of galE gene on WTAs modification may not be the same in different serotype LM strains.The results of this study provide important clues for the in-depth exploration of the pathogenic mechanism of LM.