| Trueperella pyogenes（T.pyogenes）is a Gram-positive opportunistic pathogen.In animals and humans,it often causes various septic diseases including endocarditis,liver abscess,and respiratory infections.Animal infections caused by T.pyogenes result in serious losses to the agricultural economy.TatD（twin-arginine protein translocase protein D）is a nuclease that widely present in a variety of organisms and has been reported to be involved in the immune evasion of pathogens and directly affect their pathogenicity.However,the function and inhibitors of TatD DNases in T.pyogenes have not been reported.We previously found that luteolin has an effective antibacterial activity on T.pyogenes and can inhibit the activity of enzymes such as topoisomerase and transposase.This study will investigate the function of TatD proteins in T.pyogenes and verify whether they correlate with bacterial virulence;meanwhile,we will analyze the effect of luteolin as a candidate inhibitor on the activity of TatD DNases.Our data could guide the development of novel antibacterial drugs for anti-virulence strategies.In this study,we analyzed the bioinformatics of TatD proteins and identified two TatD proteins in T.pyogenes.The TatD proteins are collectively referred to as TatD960 and TatD825,which have only a triosephosphate isomerase（TIM）-barrel domain and lack signal peptide or transmembrane domain.The distribution rate of tat D960 and tat D825 genes was 100%in 20 T.pyogenes strains.We applied the prokaryotic expression method to obtain the recombinant proteins and examined the hydrolytic activity of both on DNA to verify the biochemical functions of them.Polyclonal antibodies against TatD proteins were prepared and the distributed location of TatD proteins were detected using Western blot.The results showed that both TatD960 and TatD825 proteins possessed Mg2+-dependent DNase activity and were expressed in the intracelluar in T.pyogenes.For analysis of the function of TatD protein in bacteria,tat D-deficient mutants of T.pyogenes（BMH06-3Δtat D960,BMH06-3Δtat D825,BMH06-3Δtat D960Δtat D825）were constructed using homologous recombination.The growth ability,hemolytic activity,drug susceptibility,pathogenicity,and biofilm formation of the deficient strains were compared with those of the wild strain.Our data showed that the growth ability,hemolytic activity and drug susceptibility of deficient mutants were not altered,while the pathogenicity in mice was lower than that of the wild strain（p=0.0022）.The bacterial load in the spleen of mice inoculated with BMH06-3Δtat D960Δtat D825 was significantly lower than that in the spleen of the mice inoculated with the other three strains（p<0.01）.The biofilm formation,extracellular DNA（e DNA）release level and autolysis ability of tat D-deficient mutants were significantly lower than those of the wild strain（p<0.01）.We aimed to investigate whether luteolin could act as an inhibitor of TatD DNases and interfere with their expression.We determined the m RNA expression and protein expression of tat D genes in control and luteolin-treated groups by applying quantitative real-time fluorescence PCR（q PCR）and Western blot,respectively.We treated T.pyogenes by using subinhibitory concentration（1/2 MIC）of luteolin.The effects of luteolin on tat D genes expression were analyzed.The results showed that the expression of tat D960 m RNA was down-regulated in 85%of the strains and TatD960 protein expression was down-regulated in 90%of the strains.Following the same method,the expression of tat D825 m RNA was down-regulated in 80%of the strains and TatD825 protein expression was down-regulated in 85%of the strains.Our data suggest that luteolin can inhibit the expression of the tat D genes of T.pyogenes.To investigate the role of luteolin on the function of TatD DNases,the ability of TatD DNases to hydrolyze DNA was examined by agarose gel electrophoresis assay.We analyzed the effect of different concentrations of luteolin on the function of TatD DNases.The results showed that luteolin inhibited the activity of TatD DNases,and the inhibition showed a dose-dependent effect.The tertiary structure of TatD DNases were simulated using SWISS-MODEL,and the active sites of luteolin on TatD were analyzed by molecular docking.The results showed that luteolin was mainly bound to TatD DNases by hydrogen bonds and could interact with the key residue His106 of TatD960.Luteolin can interact with His164 and Asp212,which are the key residues of TatD825.The surface plasmon resonance（SPR）was applied to detect the interaction between luteolin and TatD.Molecular interaction assays showed that luteolin has an affinity of 2.272×10-4 M with TatD960 and 1.816×10-4 M with TatD825.These showed moderate bonding forces.Luteolin could fit kinetically to both proteins.The association and dissociation constants for luteolin and TatD960 were 395.8 1/Ms and 0.0025 1/s,respectively.The interactions between luteolin and TatD825 showed similar results with association and dissociation constants of 369.1 1/Ms and 0.0021 1/s,respectively.The data has shown a trend of fast association and slow dissociation.In summary,this study revealed that TatD proteins are Mg2+-dependent DNA endonucleases.They can affect bacterial virulence and are involved in biofilm formation.Luteolin can inhibit the transcription and protein expression levels of tat D genes in most of T.pyogenes.Luteolin can bind stably and specifically to TatD DNases and inhibit their activities.Our results provide new insight into the development of novel antibacterial drugs targeting TatD DNases,and also establish a stable theoretical foundation for the further development and application of luteolin. |
