Analysis Of Molecular Mechanism And Pathway Of Oxidative Stress Mediated By Campylobacter Jejuni Flagellin FlhF

Campylobacterjejuni(C.jejuni),as a microaerobic pathogen,is highly sensitive to the oxygen concentration in the environment.The spread and colonization of this bacterium depend on the oxidative stress resistance system,where flagella and their mediated motility assist bacteria in coping with environmental stress.As a key protein in flagella synthesis and assembly,the absence of flhF leads to complete loss of bacterial flagella and motility.When our laboratory conducted a preliminary study on the interaction between flhF and flagellar synthesis related genes,it was found that flhF mutation resulted in a significant change in the expression level of oxidative stress related genes(P<0.05).However,the specific phenotype and mechanism of oxidative stress are still unclear.Based on the construction of flhF mutant by previous researchers in the laboratory,this article uses H2O2 as an exogenous oxidant to analyze the impact of flhF mutant on the oxidative stress phenotype of C.jejuni using stress biology;Through transcriptome,molecular biology,protein interaction and other technologies,we can analyze the functional pathway of FlhF in the oxidative stress system,explore the new function of FlhF from a new perspective,and provide new evidence for the survival mechanism of Campylobacter.1 Analysis of oxidative stress biological phenotype of C.jejuni flhF mutantThis article optimizes the cultivation conditions of C.jejuni firstly,and controls a single variable to measure the bacterial growth curve under different cultivation conditions based on the factors affecting its growth(such as cultivation temperature,cultivation speed,selection of culture medium,etc.),and compares the growth status.The results showed that the bacteria could reach a logarithmic growth stage of OD600 nm=0.6,6 hours earlier than the growth rate in the early laboratory,within 6 hours under the improved version of Brucella broth at 42℃ and 150 rpm,and the number of viable bacteria in the stable stage could reach 1×1011 CFU/mL,an increase of 2 Lg CFU/mL compared to the pre laboratory culture conditions.The minimum inhibitory concentration(MIC)of H2O2 on logarithmic growth metaphase bacteria was determined to be 1.56 mM.Considering the differences in oxidative stress phenotypes of strains and the high-quality performance of RNA samples,0.7 MIC(1 mM)was used as the H2O2 stress concentration;The survival rate of bacteria after being treated with 1 mM H2O2 for different times was determined,and it was found that from the start of H2O2 stimulation to bacterial inactivation,the survival rate of ΔflhF was lower than that of WT.And when the bacteria were treated with 1 mM H2O2 for 30 minutes,the logarithmic survival rate of WT was 77%,the logarithmic survival rate of ΔflhF is 61.5%.There was a significant difference in the logarithmic survival rate between the ΔflhF and WT(P=0.0036)at which point.The absence of flhF leads to a significant decrease in the survival rate of bacteria under H2O2 oxidative stress conditions,and a significant decrease in oxygen tolerance,indicating that the presence of FlhF helps C jejuni improve its oxidative stress resistance.2 Analysis of FlhF mediated oxidative stress pathway and molecular mechanismRNA-Seq was used to analyze the effect of FlhF on the transcriptional expression level of oxidative stress coding genes,and to explore the FlhF mediated oxidative stress pathway in C jejuni.Under H2O2 oxidative stress conditions,compared to WT,flhF mutation resulted in significant changes in the transcriptional expression levels of 86 genes(Fold change≥ 2,P ≤0.05),mainly concentrated in pathways such as environmental stress,iron ion metabolism,biofilm formation,redox activity,DNA damage and repair,and bacterial chemotaxis.This study screened differentially expressed oxidative stress coding genes as potential targets for FlhF,and their transcriptional expression levels showed a decreasing trend,indicating that FlhF plays a positive role in the oxidative stress mechanism of C.jejuni.GST pull down verifies the interaction relationship between oxidative stress-related coding proteins and FlhF proteins,and deeply analyzes the molecular mechanism of interaction between FlhF and oxidative stress screening genes.The results indicate that CJJ81176_RS07825 protein interacts with FlhF protein.And the oxidative stress phenotype of AflhFACJJ81176_RS07825 dual gene mutant showed extremely significant differences compared to single gene mutant(P<0.05).FlhF contains three structural domains:B/N/G,CJJ81176_RS07825 protein binds to the B and N domains of FlhF,and each domain of FlhF exhibits corresponding oxidative stress phenotypes.CJJ81176_RS07825,as an iron transporter protein,helps protect bacteria from oxidative damage by influencing iron metabolism and participating in the Fenton reaction in the oxidative stress system of C.jejuni.Therefore,we believe that FlhF is compatible with CJJ81176_RS07825 protein interaction helps C.jejuni improve its oxidative stress resistance.