Identification And Characterization Of Intracellular Survival Related Genes Of Aeromonas Hydrophilia In Macrophages

Phagocytes play a pivotal role in the phagocytosis of microorganisms that finally leads to the elimination of many pathogenic microorganisms. The interaction between phagocytes and pathogens, in its simplest terms, consists of two interdependent phases. The first is the ingestion phase in which the leucocyte engulfs the particle; the other is the intracellular phase. Intracellular survival is an important factor determining virulence of bacteria. However, little is known about the intracellular interaction after phagocytosis of pathogens in aquaculture. Aeromonas hydrophilia is an opportunistic pathogen which was found in freshwater and marine systems, soil and human feces and so on. Recently, it became one of the main pathogen of freshwater culture animals. Therefore, in the present study, we examine the interactions between phagocytes and A. hydrophilia.We constructed A. hydrophilia mutant bank by the use of transposon mini-Tn10 which caused random gene inactiviation. We subsequently screened 11 mutants severely attenuated for intracellular survival from 102 mutants. A single transposon mini-Tn10 insertion was confirmed by southern blot. Flanking sequence of inserted site was amplified by thermal asymmetric interlaced PCR(TAIL-PCR). Flanking sequences were blasted on the National Center for Biotechnology Information(NCBI) webpage(http://www.ncbi.nlm.nih.gov/). The results showed that NO.10 mutant was interrupted in MerR, NO.28 mutant in GlpC, NO.62 mutant in flgE, NO.71 in AsmA, NO.78 in HDAC, NO.85 mutant in AhHr, but identify of flanking sequences of NO.9 and NO.11 mutants were not found on NCBI and flanking sequences of another three was not cloned.The functions of these genes including motility, observing flagella of Transmission electron microscopy, biofilm formation, chemotaxis, adhension to mucus, cell invasion, cytotoxicity and median lethal dose(LD50) were determined. Results demonstrated that the diameters of the bacterial halos for wild type W and mutant NO.62 were 39.0±2.0 and 10.0±1.0 mm, respectively. Therefore, mutant displayed remarkable impaired molitities. At the same time, transmission electron micrographs of A. hydrophila flg E mutant exhibited that a polar flagellum was absent. flgE mutant was notable decrease in chemotaxis to glucose, surface mucus, intestine mucus and gill mucus compared to the wildtype W. Mutant was significantly attenuated in adhension to surface mucus, intestine mucus and gill mucus. When no centrifugation after macrophages and bacteria were mixed, there is no significant difference in internalization of macrophages to wild type W and flgE mutant. The cytotoxicity of wild type W and flgE mutant to macrophages was 7.3%±0.2% and 4.2%±0.4%, respectively. The LD50 of the wild type W was 4.02×106 CFU/ml for zebra?sh. After zebrafish were injected ip with about 4.02×106 CFU wild type W or flgE mutant, zebrafish infected with wild type W all died in 24 hours and one zebrafish infected with flgE mutant survived in 48 hours, showing that flgE is a virulence of bacteria.