| Listeria monocytogenes is a foodborne intracellular pathogen widely distributed in the natural environment,with strong adaptability to environmental stress such as oxidation.Oxidative modification and regulation of proteins is one of the ways of L.monocytogenes to resist oxidative stress.This process requires disulfide bond forming protein（Dsb）to catalyze the formation of disulfide bonds to promote oxidative folding or modification of proteins.In L.monocytogenes,Yjb H was annotated as dsb A-like,and only lmo1059 was annotated as DsbG,no other Dsb proteins were encoded.Previous study of our research group proved that Yjb H functions like thioredoxin and plays a key role in the process of oxidant stress tolerance and pathogenicity.This study is aimed to explore the biological functions and underlying mechanisms of disulfide bond formation protein DsbG of L.monocytogenes in antioxidant stress and pathogenicity.1.DsbG has family-specific isomerase activity in L.monocytogenes,and C36 and C39 are the key sitesDisulfide reductase and disulfide isomerase activity were tested in vitro after the recombinant protein was successfully constructed.The results showed that the DsbG protein had strong disulfide isomerase activity,also with disulfide oxidoreductase activity.The two key cysteine sites of DsbG,C36 and C39,were mutated into serine by site-directed mutagenesis technique.The results showed that the mutations in C36 and C39 did not affect the reductase activity of DsbG protein.However,C39 mutation decreased the activity of disulfide isomerase,while C36 mutation directly led to the disappearance of activity,suggesting that C36 and C39 are the key active sites of DsbG as disulfide isomerase.2.DsbG plays a key role in oxidative stress tolerance of L.monocytogenesAbsence of dsbG in L.monocytogenes did not affect its in vitro growth and colony morphology,but impaired the bacterial motility.The transcriptional levels of flagella-associated genes such as fla A and flg B were significantly decreased than those of wild-type strain,which proved that dsbG caused decreased motility by affecting the formation of flagellar genes.The results of stress tolerance tests with different concentrations of oxidants showed that the resistance of L.monocytogenes to Cu2+and diamide was significantly reduced with dsbG deficiency.DsbG was involved in oxidative stress tolerance and was more sensitive to diamide.To further explore whether L.monocytogenes mediated oxidative stress tolerance through the enzyme activity of DsbG,we mutated the C36 and C39 of DsbG into Ser,and conducted the diamide oxidative stress tolerance test.The results showed that C39 mutant did not affect while C36 mutant weakened the oxidative stress tolerance of bacteria,suggesting that C36 was the key amino acid site involved in oxidative stress tolerance of L.monocytogenes.In order to further explore the mechanism of DsbG-mediated oxidative stress tolerance,whole-gene transcriptomic sequencing was used to analyze the transcription levels of EGD-e and DsbG deletion strain under diamide stress,and part of the sequencing results were verified by q RT-PCR.The results showed that the transcriptomic sequencing results were reliable.Transcriptomic data showed that the transcription levels of lmo0048（Agr B）,lmo0050（Agr C）and lmo0051（Agr A）in the Agr system involved in quorum sensing were significantly down-regulated,suggesting that DsbG might mediate oxidative stress tolerance through quorum sensing.At the same time,there were significant differences in gene expression of PTS system,glucose metabolism and other pathways,which indicated the direction for the following experiments.3.Studies on the function of L.monocytogenes DsbG in bacterial infection and pathogenicityBased on the above experimental results,the absence of dsbG affects the flagella formation and oxidative stress tolerance in vitro,and whether it also affects its infection and pathogenicity is unknown.Therefore,we conducted a study on the involvement of DsbG in bacterial infection and pathogenicity.The results showed that there was no difference in proliferation of L.monocytogenes in RAW264.7 macrophages after dsbG deletion.Decreased adhesion and invasion ability of Caco-2 cells were observed（*P<0.05）;In the intracellular migration test of fibroblast L929 cells,the number of plaque was significantly reduced after dsbG deletion（**P<0.01）while the diameter of plaque increased significantly（**P<0.01）,indicating that the infection ability of L.monocytogenes decreased after dsbG deletion,but the intracellular migration ability was enhanced.The results of organ proliferation and survival test in mice showed that there was no difference in the colonization ability of liver and spleen in mice of L.monocytogenes without dsbG,and there was no significant difference in the mortality of mice.Therefore,we believe that dsbG does not affect the overall pathogenicity of L.monocytogenes,but affects the intercellular mobility of L.monocytogenes.In conclusion,this study took disulfide bond formation protein DsbG of L.monocytogenes as the research object,and proved for the first time that DsbG affected the flagellar formation of L.monocytogenes in vitro and was involved in oxidative stress tolerance.Cysteine 36 and cysteine39 of DsbG are the key amino acid sites for DsbG to play the role of disulfide isomerase,and cysteine 36 is also the key site for DsbG to participate in oxidative stress tolerance in L.monocytogenes.DsbG did not affect the overall pathogenicity,but affected the intercellular mobility of L.monocytogenes.This study shed light for further understanding the specific mechanisms of flagellum formation,cysteine oxidative repair and antioxidant stress regulation of L.monocytogenes,and contributes to a deeper understanding of the molecular mechanism of environmental adaptation of important food-borne pathogens in vitro,which has important public health significance for the pollution control of food-borne pathogens. |
