Identification And Characterization Of Adhesion-associated Genes Of Aeromonas Hydrophila

Pathogenic bacteria adhesion to the host mucus represents the first crucial step in most infections.For many pathogenic bacteria,only when they adhere in the sensitive cells or mucosal surfaces through adhesins,can they produce toxins in the focus or invade into deep tissues,eventual penetrate into the tissues of the host.Recently,adhesion has been regarded as one of the virulence factors.However,most studies on bacteria adhesion focus on clinical pathogenic bacteria,hardly any fish pathogens have been studied to uncover the mechanisms of their adhesion.The characteristics of adhesion of Aeromanas hydrophila wild-type W strain was investigated by the method of modified indirect ELISA.The results indicated that the A.hydrophila W strain can adhere to the host eel skin mucus,adhesion of A.hydrophila W strain wasaffectedbydifferentenvironmentalfactorssuchasbacterial concentration,temperature,incubation time,pH,and Na⁺,Mg²⁺concentration and so on.In order to further reveal the mechanisms of A.hydrophila adhesion,a mutant library was constructed by the mini-Tn10 transposon mutagenesis system.Out of 332 individual colonies,9mutant strains with significantly attenuated adhesion abilitywas screened out in this study.PCR identification confirmed that these mutants were inserted by mini-Tn10,southern blotting analysis suggested that the mutations were caused by single tranposon insertion.The flanking sequences of tranposon in the mutant strains were amplified genomic walking.The results showed that the inserted sites of the mutant 121,45,77,196 and 261 were in the gene flgC,the gene of the CobQ/CobB/MinD/ParA family,the gene flgN,the gene of RbsR,and the gene of CydDC respectively.But no sequence was found to be homologous with the tranposon flanking sequence of the mutant 163 in NCBI.The function of the flgC gene in the process of A.hydrophila adhesion was investigated by analysis the phenotypic characteristics including movement,adhesion,and biofilm formation of the wild-type,the mutant and the complementation.The results displayed the mutant strain 44exhibited significant defect in movement,adhesion,and biofilm formation compared with the wild-type strain and the complementation,which suggested that flgC plays an important role in A.hydrophila adhesion to its host mucus.