Listeria Monocytogenes Glutathione Synthase GshF Contributes To Environmental Adaptation And Infection

Listeria monocytogenes(L.monocytogenes)is a Gram-positive bacteria.It can survive in extreme environments such as acidic,alkaline,high osmotic pressure,low temperature and other environments.The strong viability of Listeria is attributed to its antioxidant system.Listeria has two sets of antioxidant systems:Thioredoxin(Trx)and Glutaredoxin(Grx)systems.Glutathione synthase GshF(encoded by the lmo2770)is one of the important components in the glutredoxin system and plays a vital role in the antioxidant process,while the specific mechanism of the role of GshF in Listeria is not yet clear.Therefore,in this study,the molecular biology,cell biology,stress biology and other methods were used to explore the mechanism of GshF in the process of Listeria anti-oxidative stress,flagella formation and infection.The results of the study are as follows:the gshF mutant strain were constructed by the method of homologous recombination,and on this basis,to construct complemented strains.Then we studied the anti-oxidative stress ability,flagella formation ability and the ability to infect cells of the gshF mutant strain.Deletion of gshF did not affect bacterial in vitro growth,but the flagellar motility of the gshF mutant strain was lower than wild-type in a low agar motility assay.Furthermore,Western blot and q RT-PCR assays were performed using a gshF mutant strain to determine the effect of gshF on flagella formation.The results suggested that the lack of gshF affect the transcription and translation of flagellar genes,such as gma R,fla A,mot B,fli M,fli F,flg B,flg E,flg G,fli E,flh A,flh B,flg D,flg E,flg K,and flg L.Among them,the transcription and translation levels of gma R,fla A gene were significantly reduced.To determine whether gshF could contribute to oxidative tolerance,the EGD-e,?gshF and C?gshF were exposed to different oxidative agents which including H₂O₂,diamide and copper.The EGD-e and the mutants appeared the similar resistance to hydrogen peroxide under concentration of 10 m M.And mutation of gshF rendered this bacterium less resistant to copper and diamide.We attempted to further explore the functions of gshF during the bacterial intracellular infection.The gshF mutant strain showed the same efficiency to proliferate inside the macrophages J774 and RAW cells post infection compared with EGD-e.In the epithelial cells Caco-2,the gshF mutant strain was significantly more invasive,compared to that of the wild-type and complement strains,but did not affect bacterial adhesion to cells.We found that the gshF mutant strain has the same ability to form plaques on L929 fibroblast monolayers compared to that of EGD-e,suggesting that the ability of this bacterium to spread among cells was not affected by this gene.And also the gshF mutant strain exhibited less efficient proliferation and recovery in the spleens and livers of the infected mice.Then we did the transcriptome analysis of EGD-e and gshF mutant strain treated with 2 m M diamide.Transcriptome sequencing results showed that the PTS system-related genes:lmo1997(ii A),lmo2000(ii D),lmo2001(ii C),lmo2002;Gnt R family green-regulating factor genes:lmo2003,lmo2004;and other genes lmo1998,lmo1999 in gshF mutant strain had significantly higher transcription levels than that of wild strains.Using q RT-PCR and fluorescent reporter system to verify the transcriptome data,relevant experimental results indicated that gshF may participate in the process of Listeria monocytogenes anti-oxidative stress and cell infection by regulating related genes.In summary,this study took GshF as the research object,and explored the role of GshF in the process of Listeria in resisting oxidative stress,flagella formation and host infection.This study lays a foundation for in-depth exploration of the specific mechanisms of the biological processes of Listeria against oxidative stress,flagella formation and cell infection,and is of great significance in preventing and controlling Listeria infection and protecting public health.