Molecular Evolution,pathogenicity And Related Mechanisms Of Moraxella Catarrhalis With Different Drug Resistance Phenotypes

Objective:To explore the molecular background of important colonies of Moraxella catarrhalis;To elucidate the pathogenicity of M.catarrhalis with different drug resistance phenotypes;To investigate the possible mechanism of M.catarrhalis adhesion and invasion into A549 alveolar epithelial cells.Methods:(1)Based on MALDI-TOF MS identification,in vitro drug sensitivity an d MLST typing,70 clinical isolates covering CC363,CC449,CC224 and CC446 were screened.For 70 strains to be tested,PFGE typing,copB typing,16S rRNA typing,LOS serotype molecular typing,high-throughput sequencing and bioinformatics analysis were carried out to evaluate the resolution and accuracy of common molecular typing methods.(2)Based on 46 virulence genes,19 strains with different virulence gene combinations were selected to compare their ability to adhere to and invade A549 cells in vitro.The lung clearance rate,pathological changes of lung tissue and the production of cytokines were analyzed by constructing a mouse lung infection model.Two strains of M.catarrhalis with significantly different virulence and drug resistance phenotype were selected to infect A549 cells,and the mRNA of A549 cells was extracted for transcriptome analysis.(3)For TLR2 receptor,TLR2 agonists and inhibitors were used to construct siRNA TLR2 A549 cells and TLR2-/A549 cells,and then the role of TLR2 receptor in the process of adhesion and invasion of M.catarrhalis to A549 cells was studied through adhesion test,invasion test,transmission electron microscopy and other methods;Then,the lung infection model of TLR2-/-C57/BL6J mice was established for in vivo validation.The possible mechanism of actin polymerization/depolymerization regulation system internalizing M.catarrhalis in A549 cells through macropinocytosis was studied by transmission electron microscopy,F-actin/G-actin ratio analysis,and high content screening.Immunoblotting was used to detect fibronectin-α5β1 integrin pathway and TLR2-PI3K-AKT signal pathway were used to screen which related proteins might participate in the process of adhesion and invasion of M.catarrhalis to A549 cells.Results:(1)WGST constructed a highly precise typing system using SNPs.PFGE showed high consistency and high resolution with WGST in the same clone or clones with close genetic relationship,while MLST had very limited resolution.CopB Ⅱ is highly related to CC449.LOS B type is mainly concentrated in CC224,and 70 isolates belong to 16S rRNA type I related to increasing pathogenic potential and serum resistance.The screening results of virulence genes of M.catarrhalis showed that there were significant differences among the four clonal complexes,the macrolide resistant group and the sensitive group,among uspA2,uspA2H,pilO,lbpB,lexl,modM,mboIA and mboIB.(2)Macrolide sensitive isolates have weak adhesive ability but strong invasive ability,while macrolide resistant isolates have strong adhesive ability but weak invasive ability.The adhesive and invasive abilities of different clonal complexes were also significantly different.(3)The adhesion and invasion of 73-OR to TLR2-/-A549 cells was greatly reduced.Meanwhile,TLR2-/mice with TLR2 absent,which led to the decreased adhesion of 73-OR to lung tissue,significantly accelerated lung clearance,and the local inflammatory response was also milder than that of normal mice after infection.M.catarrhalis invades A549 cells in the form of macropinocytosis,regulation of actin polymerization/depolymerization and fibronectin-α5β1 integrin system and TLR2-PI3K-AKT signal pathway may be involved in the process of M.catarrhalis adhesion and invasion of A549 cells.Conclusions:(1)WGST constructed a highly precise typing system using SNPs,which greatly deepened the understanding of the origin and evolution of macrolide resistant M.catarrhalis in this study;Macrolide resistant clones have obvious "fitness cost" phenomenon,which is mainly manifested by slow growth and reduced virulence.(2)The ability of M.catarrhalis with different macrolide resistant phenotypes to adhere to and invade A549 cells varies greatly.Sensitive isolates have weak adhesion but strong invasion,while drug-resistant isolates have strong adhesion but weak invasion.(3)Fibronectin-α5β1 integrin system and TLR2 PI3K-AKT signal pathway may participate in the process of adhesion and invasion of M.catarrhalis to A549 cells through the regulation of actin polymerization/depolymerization.