Mechanism Of CpxRA Two-component System In Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)belongs to Extraintestinal pathogenic Escherichia coli(Ex PEC),which is a gram-negative,facultative anaerobic microorganism.APEC is one of the common enteral infectious pathogens of poultry,which can cause local or systemic infections accompanied by multiple organ damage disease syndrome in chickens,ducks,geese and other birds.The increasing morbidity and mortality of birds means huge economic losses.Similar to Uropathogenic Escherichia coli(UPEC)and Neonatal Meningitis Escherichia coli(NMEC),APEC could be transmitted to humans through foodborne cross infection by handling live poultry or eating undercooked poultry products,indicating a zoonotic risk and a potential danger to human health.Until now,there has been no better strategies to control colibacillosis in poultry than to use large amounts of antibiotics.However,the long-term use and abuse of antibiotics has led to the emergence of multi-drug resistant strains.The expression of efflux pump gene is a common mechanism of multidrug resistance in bacteria.Two-component regulatory system(TCS)is a regulatory system widely distributed in a variety of bacteria.It has been reported that CpxRA two-component system has an effect on bacterial drug resistance.CpxRA is involved in bacterial viability and pathogenicity,including virulence,antioxidant stress,etc.However,the specific regulatory mechanism of CpxRA two-component system remains unclear.This study revealed the effect of CpxRA two-component system on antibiotic sensitivity and biofilm formation,as well as the regulatory mechanism of the Emr KY efflux pump,and further indicated the important regulatory role of CpxRA two-component system in APEC under antibiotic stress.1.In order to confirm whether the deletion of cpxRA gene has an effect on the growth of APEC strain.We conducted growth curve test and viable count test(CFU).The growth trend of each strain was measured under the same culture conditions.The results showed that the growth trends of wild WT/p STV28,mutant XM1/p STV28 and replacement XM1/p CcpxRA were similar in LB medium containing 15μg/m L Cm.There was no significant difference in CFU in the lag period,exponential period and stationary period of the growth curve,indicating that the absence of cpxRA had no effect on the growth of APEC strain.2.The effect of cpxRA on APEC biofilm formation was tested by biofilm formation test.The results showed that,compared with the wild strains,the mutant strains had reduced biofilm formation,while the completed strains recovered the biofilm formation ability.The drug sensitivity test was conducted to detect the sensitivity to erythromycin and ofloxacin of APEC,and the results showed that the sensitivity of APEC40 to the above two antibiotics significantly decreased in the wild strain.The transcriptional levels of genes emr K and emr Y,encoding multi-drug efflux pump,were detected by RNA extraction,reverse transcription PCR and real-time quantitative fluorescence PCR(RT-q PCR).The results showed that the transcriptional levels of emr K and emr Y in the mutant were improved compared with those in the wild strain,while the transcriptional levels of these two genes in the complement strain were recovered to similar levels to those of the wild type.These data suggested that Cpx signaling plays an important regulatory role in the biofilm formation of APEC and that CpxR negatively regulates the susceptibility of bacteria to ofloxacin and erythromycin.3.The binding of CpxR protein to the promoter region of emr KY was investigated by molecular biological methods including protein expression and purification technology in vitro and electrophoretic mobility change test(EMSA).The positive control showed that CpxR could bind to its own promoter.The results indicated that CpxR protein could directly bind to the promoter region of emr KY gene,and the bandshifting intensity increased with the increase of CpxR concentration.The results demonstrated that CpxR can specifically bind to the emrKY promoter,indicating that CpxR can directly regulate the transcription of emrKY efflux pump.