Mechanical Study Of Thioredoxin A Involved In Listeria Monocytogenes Infection

Listeria monocytogenes is a Gram-positive,food-borne intracellular pathogen,which can cross the host's blood-brain barrier,placental barrier,causing meningitis,abortion and other listeriosis with up to 30%lethality.Listeria monocytogenes requires a series of virulence factors for invasion,thereby destroying the host's immune defense and stress systems.Thioredoxin(Trx)is a class of small redox proteins known to be present in all organisms.Trx acts as an antioxidant by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange.It is still unknown how L.monocytogenes utilizes Trx to maintain its intracellular –SH homeostasis under various oxidant stresses conditions.Therefore,in this study,we aimed to explore the molecular mechanism of Trx A,by which to regulate gene express under oxidative stresses;to demonstrate the modification mechanism mediated by Trx A to virulence factors of bacteria;demonstrate the redox interactions between Trx A and flagellar protein Mog R/Gma R as well.Phenotype and in vivo/vitro assays were performed to reveal the biological functions of Trx A.The study showed that expression of L.monocytogenes Trx A was significantly induced when bacteria subjected to the thiol-specific oxidizing agent diamide(P<0.001),and deletion of trx A markedly compromised its tolerance to diamide,and subsequently impaired its survival inside human intestinal epithelial cell Caco-2(P<0.001).However,either deletion or constitutive over expression of Trx A severely attenuated this pathogen in mice(P<0.001).EMSA assay showed that the expression of Trx A was regulated by the transcriptional factor Sig H,which bound to the promoter region of trx A in vitro.Transcriptome analysis of L.monocytogenes revealed over 270 genes were differentially expressed in the ?trx A mutant by contrast to its parent strain.Most strikingly,the virulence genes plc A,mpl,act A,and plc B showed significantly higher expression in wild-type compared to the ?trx A(EGD-e/?trx A?2).The ITC assay showed that Trx A and the regular protein Prf A interacted in an exothermic manner,involving a broken of ionic bonds(K=4.00E±1.13E4M-1).Interestingly,deletion of trx A completely compromised formation offlagella,resulting a nonmotile bacterium.We further showed by ITC assay that Trx A interacted with Mog R,a flagellar synthesis protein,by using chemical energy(K=1.28E±1.56E4M-1).In conclusion,we here,for the first time,demonstrate that Listeria monocytogenes Trx A is involved in the antioxidant stresses by disulfide bonds shuttling system,maintaining intracellular–SH homeostasis.This study sheds light on a novel mechanism used by Listeria monocytogenes and other intracellular Gram-positive bacteria to regulate flagella formation,virulence gene expression and survival under oxidant stress conditions.