The Role And Mechanism Of Twin-arginine Translocation System In Brucella Melitensis Virulence

Brucella,the causative agent of brucellosis,is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals,including humans.Brucellosis causes serious economic losses to animal husbandary,as well as other serious public problems.Compared with other bacterial pathogens,no typical virulence factors such as toxin,adhesion factor and Fe ionophore were found in Brucella.Brucella virulence is more reflected in its interaction with the host,and the secretion system plays a key role in the process of Brucella-host interaction.Although Brucella genome may encode multiple secretion systems,only the structure and function of type IV secretion system(T4SS)are intensively studied.Therefore,the research on and the understanding of Brucella secretion system is helpful to understand the pathogenic mechanism of Brucella.The twin-arginine translocation(Tat)pathway transports folded proteins across the cytoplasmic membrane and has been implicated in virulence in many bacterial pathogens.However,the roles of the Tat system and related substrates in Brucella remain unclear.We report here that disruption of Tat increases the sensitivity of B.melitensis M28 to the membrane stressor sodium dodecyl sulfate(SDS),indicating cell envelope defects,as well as to EDTA.In addition,mutating Tat renders M28 bacteria more sensitive to oxidative stress caused by H₂O₂.Further,loss of Tat significantly attenuates B.melitensis infection in murine macrophages ex vivo.Using a mouse model for persistent infection,we demonstrate that Tat is required for full virulence of B.melitensis M28.Genome-wide in silico prediction combined with an in vivo amidase reporter assay indicates that at least 23 proteins are authentic Tat substrates,and they are functionally categorized into solute-binding proteins,oxidoreductases,cell envelope biosynthesis enzymes,and others.A comprehensive deletion study revealed that 6 substrates contribute significantly to Brucella virulence,including an L,D-transpeptidase,an ABC transporter solute-binding protein,and a methionine sulfoxide reductase.Collectively,our work establishes that the Tat pathway plays a critical role in Brucella virulence.Tat pathway transports folded proteins across the cytoplasmic membrane;protein substrates translocated by Brucella include ABC transporters,oxidoreductases,and cell envelope biosynthesis proteins.Previously,we showed that a Tat mutant of B.melitensis M28 exhibits reduced survival within murine macrophages.In this study,we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo.We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants.A total of 6085 macrophage proteins were identified with high confidence,and 79,50,and 99 proteins were differentially produced upon infection with the Tat mutant at 4,24,and 48 hpi,respectively,relative to the wild-type infection.Gene ontology and KEGG enrichment analysis indicated that immune response-related proteins were enriched among the upregulated proteins.Compared to the wild-type M28 infection,the most upregulated proteins upon Tat-mutant infection included the cytosolic nucleic acid signaling pathway-related proteins IFIH1,DHX58,IFI202,IFI204,and ISG15 and the NF-?B signaling pathway-related proteins PTGS2,CD40,and TRAF1,suggesting that the host increases the production of these proteins in response to Tat mutant infection.Upregulation of some proteins was further verified by a parallel reaction monitoring(PRM)assay.ELISA and q RT-PCR assays indicated that Tat mutant infection significantly induced proinflammatory cytokine(TNF-?and IL-6)and nitric oxide(NO)production.Finally,we showed that the Tat mutant displays higher sensitivity to nitrosative stress than the wild type and that treatment with the NO synthase inhibitor L-NMMA significantly increases the intracellular survival of the Tat mutant,indicating that NO production contributes to restricting Tat mutant survival within macrophages.Collectively,this work improves our understanding of host immune responses to Tat mutants and provides insights into the mechanisms underlying the attenuated virulence of Tat mutants.The virulence of the Tat mutant of Brucella was significantly reduced in murine macrophages and mouse model.In order to explore whether the attenuated strain has application potential,we assessed the immunoprotective ability of the Tat mutant in mouse model.Vaccination of BALB/c mice with the Tat mutant conferred significant protection against the wild-type M28 challenge to the levels that are comparable to that induced by a licensed vaccine M5-90.ELISA and q RT-PCR assays showed that Tat mutant can significantly induce the production of IFN-?and Brucella-specific Ig G in mice.In conclusion,Tat pathway plays a critical role in Brucella virulence.Our work has opened a new avenue for understanding Brucella pathobiology and virulence strategies.The severe virulence defects caused by Tat deficiency highlight that the Tat system may serve as a potential anti-brucella target for developing antimicrobials or live attenuated vaccines.Future work should be directed toward identifying more Tat substrates and defining the functions of the substrates.