CpxAR Two-Component System Affects Biofilm Formation And Virulence In Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae(APP)is a highly contagious respiratory pathogen that causes porcine pleuropneumonia,known as fibrino hemorrhagic necrotizing broncho pneumonia and fibrinous pleuritis,and leads to great economic losses in the swine industry worldwide.Previous studies have demonstrated that Cpx AR two-component regulatory system was involved in virulence,biofilm formation and antibiotic resistance in many other bacteria,while its function in APP remains unclear.This paper aims to investigate the role of Cpx AR in virulence,biofilm formation and antibiotic resistance in APP through a gene deletion strain.1.Identification of ΔCpx AR and growth curvesMutant strain ΔCpx AR and complemented strain CΔCpx AR were verified by PCR.Growth curves of different strains in two culture media was monitored by OD600.Stringent response assays showed that Cpx AR played a crucial role in cell envelope stress in APP.2.Comparison of biofilm formation and antibiotic resistance between wild type strain and ΔCpx ARWe compared biofilm-forming ability between wild type strain and ΔCpx AR through a crystal violet staining in 96-well plates.The results demonstrated that cpx AR mutant ΔCpx AR showed a marked reduction in the amount of biofilm compared to wild type strain.In this study,we also investigated the role of cpx AR in the antibiotic resistance of APP by comparing minimum inhibitory concentration(MIC).3.Molecular mechanism of biofilm formation in APP by Cpx ARSeveral identified biofilm-related genes showed differential expression by q PCR.An electrophoretic mobility shift assay and DNaseⅠfootprinting assay indicated that Cpx AR can directly regulate the expression of rpo E,but not hns,to affect the function of the pga operon,which synthesis component of biofilm in APP.4.Comparison of virulence between wild type strain and ΔCpx ARThrough a survival curve method and bacterial load assay,we demonstrated that Cpx AR inactivation impairs virulence and colonization ability of APP.