The Roles Of Hr And IlvB-1 Gene On Aeromonas Hydrophilia Survival In Macrophages

Intracellular survival is an important process for obligate intracellular pathogens to initiate infection.It has been confirmed that obligate intracellular paththogenic bacteria have developed a series of strategies to resist killing of phagocytes and survive for further spread.Recent researches suggested that not only can obligate intacellular pathogens survive in phagocytes,but some pathogens that were generally seen as non-obligate intacellular pathogens can also survive in macrophages.Survival in phagocytes has been regarded as an important virulence factor of bacteria.However,the researches on the intracellular survival of non-obligate intacellular pathogens was almost limited to staphylococcus aureus,seldom researches on survival in phagocytes of other non-obligate pathogens t were reported.In our previous researches,we found that Aeromonas hydrophila,a wildspread pathogen of aquatic animals can also survive in macrophages of Anguila japonica for at least 24 h,but the researches on its mechanism just started.In this study,we further investigated the molecular mechanisms of A.hydrophila to survive in host macrophages.At first,a mutant library of impaired intracellular survival bacteria was constructed by the Mini-Tn10 transposon mutagenesis system,2 mutant strains M85 and M159 with significantly attenuated intracellular survival ability were screened out in this study.PCR identification and Southern blot analysis suggested that these mutants were inserted by single transposon.To abtain the full-length of target gene,the flanking fragments of inserted transponson were amplified by TAIL-PCR,and then sequenced and spliced.Via online BLAST,it suggested that the inserted site of M85 mutant was Hr gene.This ORF encodes a 183-amino acid protein and displays the highest sequence identity(98%)with the hemerythrin(Hr)protein of A.hydrophila ATCC 7966.The inserted site of M159 mutant was ilvB-1gene.This ORF encodes a 573-amino acid protein and displays the highest sequence identity(98%)with the Acetolactate Synthase(ilvB-1)protein of A.hydrophila YL17.In order to further investigate the function of these two genes on the process of bacterial intracellular survival,the recombinant expression plasmids pACYC184-Hr and pACYC184-ilvB-1 were constructed.And these two recombinant plasmids were transformed into M85 and M159 respectively to construct complementary strains C85 and C159 by Electroporation.The proteins expression in the complementary strains was identified by Western blot.Intracellular survival,motility,hydrogen peroxide sensitivity and biofilm formation of the wild-type,the mutants and the complementary strains were evaluated.The results suggested that Hr gene worked as the following to help A.hydrophila survival in macrophages:(1)acts as oxygen sensor and responds to change of oxygen concentration,which will lead to change of respiration and other metabolic efficiency;(2)resists respiratory burst through repairing iron sulfur protein clusters to eliminate ROS.The roles of ilvB-1gene in A.hydrophila survival in macrophages were speculated to involve in synthesis of branched chain amino acids which were essential for bacteria growth and maintaining in severe intracellular environment.