Mechanism Of Listeria Monocytogenes Glutaredoxin-mediated Bacterial Oxidative Tolerance And Infection

Listeria monocytogenes(L.monocytogenes)is a Gram-positive intracellular parasite that can cause foodborne zoonotic diseases.This pathogen can survive in extreme environments such as oxidative stress,and the antioxidant property is indispensable for the host infection.Thioredoxin(Trx)system and Glutaredoxin(Grx)system are the two important thiol-dependent reductase systems in the redox balance in bacteria.It is known that the Trx system is functional in maintaining reducing environment of cytosol in L.monocytogenes,whereas it is still unknown whether Grx system exists or not.L.monocytogenes has been shown to encode a putative glutaredoxin(encoded by lmo2344),which contains a conserved CXXC active motif,while the underlying roles remain unknown.In the current study,we aimed to elucidate the molecular functions and underlying mechanisms of the L.monocytogenes Grx system,with a view to determine whether it contributes to biological processes related to bacterial anti-oxidative stress and infection.Here we suggest an unexpected role of L.monocytogenes Grx in oxidative tolerance and intracellular infection.The recombinant Grx was able to efficiently catalyze the thiol-disulfide oxidoreduction of insulin in the presence of DTT as an election donor.Deletion of grx did not affect bacterial in vitro growth,but significantly decreased the bacterial colony size.Unexpectedly,the deletion of grx resulted in a remarkably increased tolerance and survival ability of this bacteria when exposed to various oxidizing agents,including Cu2+,Cd2+ and diamide.Furthermore,loss of grx significantly promoted bacterial invasion and proliferation in intestinal epithelial Caco-2 cells and J774 A.1 macrophages,as well as a notably increasing invasion but not cell-to-cell spread in the murine fibroblasts L929 cells.More importantly,L.monocytogenes lacking the glutaredoxin exhibited more efficient proliferation and recovery in the spleens and livers of the infected mice,and hence became more virulent.The transcriptome analysis of L.monocytogenes EGD-e and grx-deficient strain treated with 4 m M diamide revealed that oxidative tolerance-related genes(lmo0722,qox A,qox B and gr)and virulence-related genes(inl A,inl B,opu CA,opu CB,opu CC and opu CD)were significantly up-regulated in grx-deficient strain compared to wild-type EGD-e.Real-time quantitative PCR,fluorescence report system and Western Blot were used to verify transcriptome data.These results intuitively indicated that L.monocytogenes Grx is involved in bacterial oxidative resistance and virulence regulation.Additionally,the results of electrophoretic mobility shift assay(EMSA)showed that Grx indirectly regulated lmo0722,qox A,qox B,gr,inl A,inl B,opu CA,opu CB,opu CC and opu CD genes.In summary,we here for the first time demonstrated that L.monocytogenes glutaredoxin plays a counterintuitive role in bacterial oxidative resistance and intracellular infection,which is the first report to provide valuable evidence for the role of glutaredoxins in bacterial infection.More importantly,this study suggests a favorable model to illustrate the functional diversity of zoonotic bacterial Grx systems during anti-stress survival and host infection as well as the relationship between bacterial redox proteins and pathogenicity.