Mechanisms Of The Putative Thioredoxin YjbH From Listeria Monocytogenes Involved In Oxidative Tolerance And Bacterial Infection

Listeria monocytogenes is an intracellular facultative bacterial pathogen that can cause serious infections leading to high mortality in the immunocompromised individuals and pregnant women.This pathogen is well-adapted to various stress environments,especially employing various strategies to counteract oxidative conditions present in the environment and within the vacuolar compartment of phagocytic cells.The thioredoxin(TrxA)family,containing a highly conserved Cys-Xaa-Xaa-Cys motif,is widely distributed in bacterial species and has been determined to play an important role in cellular defense against oxidative stress.In L.monocytogenes,our previous work has collectively elucidated that TrxA as a vital cellular reductase is essential for maintaining a highly reducing environment in the bacterial cytosol,which provides a favorable condition for protein correct-folding,and therefore contributes to bacterial antioxidant and virulence.However,the biological roles and the underlying mechanisms of the putative thioredoxin YjbH from this pathogen has remained unknown.Here we aimed to fully illustrate the molecular functions of L.monocytogenes YjbH,with a view to determine whether it contributes to the biological processes related to bacterial oxidative tolerance and host infection.Specifically,we found that lack of YjbH had no influence on bacterial growth in vitro,while significantly decreased the colony size by using the stereo and transmission electron microscopy observation.YjbH could be markedly induced in bacteria treated with the oxidizing agents,and moreover,deletion of this protein totally compromised tolerance of the pathogen against the oxidative oxidants,copper and cadmium.More importantly,YjbH could directly interact with SpxA1,an ArsC family transcriptional regulator similar to the disulfide stress regulator Spx conserved in Firmicutes,suggesting that YjbH contributes to defend against oxidative stress most probably via the interaction with SpxA1.In addition,the absence of YjbH strongly attenuated the ability of bacterial invasion and intracellular growth in human intestinal epithelial cells and murine macrophages,as well as cell-to-cell spreading in murine fibroblast cells.Furthermore,YjbH was found to essentially contribute to survival and proliferation in mouse organs(livers and spleens),and therefore was required for bacterial virulence.Transcriptome analysis of L.monocytogenes treated with 0.25 mM cadmium revealed that the virulence-associated genes,hly,actA,mpl,inlC,plcA/B,were significantly down-transcribed in the YjbH deletion mutant,which was further validated by the RT-PCR method.To our surprise,the transcript of these virulenceassociated genes was not affected by deletion of YjbH when bacteria grown in BHI medium,but expression of PrfA was strongly induced under the same conditions.Interestingly,we found that YjbH was highly involved in the positive-regulation of the phosphotransferase(PTS)system,the major carbohydrate transport system that catalyzes the phosphorylation of incoming sugar substrates concomitantly with their translocation across the cell membrane in bacteria.Collectively,these data indicated that YjbH participates in regulating the key virulence genes with a complicated regulatory network involving PrfA and PTS system,and thereby contributes to bacterial host infection and pathogenicity.In summary,we here for the first time revealed the biological roles and the underlying regulatory mechanisms of the putative thioredoxin YjbH in the oxidative stress tolerance and intracellular infection of the foodborne pathogen L.monocytogenes.The data from our study provides a valuable model for clarifying the pathways associated with the potential roles of thioredoxins from foodborne pathogens in improving survival in the external environment,and more importantly,successfully establishing infection within the host.