The Potential Evaluation Of TolC As A Target For Drug Design In Riemerella Anatipestifer

Riemerella anatipestifer infection causes infectious serositis in ducks,resulting in significant economic losses to the duck industry.Due to the large number of RA serotypes and no cross protection between them,antibiotics are still commonly used to prevent and control the infection of this bacterium.However,the bacteria had resistance to antimicrobial drugs and the resistance spectrum showed a trend of expanding.Therefore,finding new drug targets is the premise of developing new drugs.The goal of this study is to evaluate the potential of two TolC proteins(TolCA and TolCB)in the RA-CH-1 strain as antimicrobial design targets.The main experimental results are as follows:1.TolCA participated in the tolerance of gentamicin and SDS in RA-CH-1Sequence analysis showed that there were two genes encoding TolC protein in the genome of RA-CH-1 strain,namely B739_RS04270 and B739_RS08830,which were renamed tolCA and tolCB,respectively.In order to study the role of TolCA and TolCB in drug resistance,tolCA deletion mutant,tolCB deletion mutant,and tolCAtolCB double deletion mutant were constructed in this study.MIC assay results showed that tolCA deletion was significantly more sensitive to gentamicin and detergent SDS than parent strain.There was no significant difference in the sensitivity of tolCB deletion bacteria to all tested antibiotics compared with the parent strain.These results indicated that TolCA was involved in the tolerance of RA-CH-1 to gentamicin and SDS,while TolCB was not involved.2.TolCA participates in the disinfectant resistance of RA-CH-1 strainIt has been shown that TolC plays a key role in efflux disinfectants.Therefore,the sensitivity of tolCA and tolCB deletion strains to three commonly used disinfectants was tested in this study.The results showed that,compared with the parent strain,the sensitivity of tolCA deletion bacteria to benzalammonium bromide and sodium hypochlorite was significantly enhanced,but the sensitivity to hydrogen peroxide was not significantly different.There was no significant difference in the sensitivity of tolCB deletion bacteria to the three disinfectants compared with the parent strain.In conclusion,TolCA is involved in RA-CH-1 tolerance to benzalammonium bromide and sodium hypochlorite,while TolCB is not involved.3.TolCA,TolCB participate in metal efflux of RA-CH-1TolC protein is an important component of RND efflux pump,and RND efflux pump is involved in metal efflux as well as drug resistance.In order to explore whether TolCA and TolCB participate in metal efflux,the sensitivity of single and double mutant strains of tolCA and tolCB to MnCl2,ZnSO4,CuCl2,FeSO4,CoCl2 and NiCl2 was tested.The results showed that the sensitivity of RA-CH-1 to MnCl2,ZnSO4,CuCl2,FeSO4,CoCl2 and NiCl2 was significantly enhanced after tolCA knockout,while the sensitivity to MnCl2 was significantly enhanced after tolCB knockout.The intracellular metal content of Mn(Ⅱ),Zn(Ⅱ),Cu(Ⅱ),Fe(Ⅱ),Co(Ⅱ)and Ni(Ⅱ)was increased in RA-CH-1ΔtolCA strain,and the content of Mn(Ⅱ)was increased in RA-CH-1ΔtolCB strain compared with the parent strain when the bacteria were cultured in TSB containg corresponding metal.In conclusion,TolCA is involved in the efflux of Mn(Ⅱ),Zn(Ⅱ),Cu(Ⅱ),Fe(Ⅱ),Co(Ⅱ)and Ni(Ⅱ),while TolCB is only involved in the efflux of Mn(Ⅱ)in RA-CH-1.4.The combination of gentamicin with efflux pump inhibitor can enhance the bactericidal effect of gentamicinGentamicin is a commonly used antibacterial drug.In order to further evaluate whether TolCA can be used as a design target for antibacterial drugs,the bactericidal effect of GEN combined with RND efflux pump inhibitor CCCP was determined in this study.The results showed that CCCP can improve the bactericidal effect of GEN against RA-CH-1,and can be used to improve the bactericidal efficiency of antibiotics.5.TolCA was involved in the pathogenesis of RA-CH-1 to ducklingsCompared with the parent strain,tolCA deletion strain significantly reduced the mortality of ducklings after infection,and the colonization levels in blood,liver,spleen and brain of ducklings were significantly decreased,suggesting that tolCA knockout affected the virulence of RA-CH-1 to ducklings.tolCB knockout did not affect the virulence and colonization ability of ducklings.Taken together,it shows that TolCA can be used as a target for drug design.