The Potential Evaluation Of A RND Efflux Pumps Encoded By B739＿RS08835 As A Drug Design Target In Riemerella Anatipestifer

Riemerella anatipestifer（R.anatipestifer,RA）is a Gram-negative bacterium.As the pathogen of infectious serositis,R.anatipestifer is one of the causes of duckling mortality,which seriously threatens the healthy development of China’s duck industry.Due to the large number of serotypes,lack of cross-protection between serotypes and resistance to multiple drugs,the effectiveness of vaccination or drug treatment to prevent the disease is limited.Therefore,in order to solve the difficult prevention and control of the disease,the study for effective drug targets has become urgent.This study aims to investigate whether the RND efflux pump encoded by B739＿RS08835 is involved in the efflux of drugs or metal ions,and is involved in the resistance to disinfectant sodium hypochlorite（NaOCl）,in order to evaluate whether it can be used as a target for drug design.The main results are as follows:1.Construction of B739＿RS08835 markerless deletion mutants in R.anatipestifer CH-1and identify the function in metal effluxThe RND efflux pump is divided into two subfamilies,one involved in drug resistance and the other involved in effluxing metal ions.To identify whether B739＿RS08835 is involved in the efflux of metals,the markerless deletion strain and its complement strain of B739＿RS08835 were constructed in the R.anatipestifer CH-1 strain.Sensitivity of CH-1,CH-1ΔB739＿RS08835 and CH-1ΔB739＿RS08835^Cto Mn Cl₂,Zn SO₄,Cu Cl₂,Co Cl₂ and Ni Cl₂was determined.The results showed that zone of inhibition of the B739＿RS08835 deletion strain was significantly increased under Mn Cl₂ treatment compared with the parent strain.Consistently,the growth of CH-1ΔB739＿RS08835 in liquid medium with manganese was strongly inhibited in comparison to the parental strain.The content of intracellular metal was detected by ICP-MS,the results showed that the content of intracellular manganese in CH-1ΔB739＿RS08835 strain was higher than parent strain.In conclusion,it shows that the RND efflux pump encoded by this gene is specifically involved in the efflux of manganese.Based on the above results,B739＿RS08835 was named as manganese efflux transporter B（MetB）.2.MetB is involved in NaOCl resistance.Studies have shown that the sensitivity of bacteria to the disinfectant NaOCl can be increased by the accumulation of intracellular manganese.Thus,this study further investigated the survival rate of CH-1Δmet B after NaOCl treatment was determined.The results showed that the survival rate of CH-1Δmet B was lower than parent strain,indicating that MetB was involved in resistance to disinfectant NaOCl killing.To demonstrate that met B is regulated by NaOCl,the expression of met B after NaOCl treatment was detected by q RT-PCR.It was found that the transcript level of met B in the parent strain was significantly up-regulated compared with the untreated group,which further verified that the presence of MetB can sense the stimulation of NaOCl and protect itself from being killed.3.The combination of efflux pump inhibitors and NaOCl can enhance its bactericidal effect.NaOCl is a commonly used disinfectant in practice.In order to further evaluate whether MetB can be used as a target for new antimicrobial drug design,this study accessed the killing effect of RND efflux pump inhibitors combined with NaOCl.The results showed that the survival rate of the combined treatment group was significantly lower than the single treatment group,indicating that the combination of the two treatments could improve sterilization efficiency.4.Drug susceptibility testing of met BThe sensitivity of met B markerless deletion mutants to 14 drugs in 9 categories was detected by minimum inhibitory concentration（MIC）.The results showed that there was no significant difference in the sensitivity of B739＿RS08835 deletion strain to drugs compared with the parent strain,indicating that it was not involved in drug resistance.Protein sequence analysis showed that the homologous proteins of MetB were mainly distributed in the members of the Weeksellaceae.In conclusion,the RND efflux pump encoded by B739＿RS08835 can be used as a target for drug design.