Construction And Characterization Of OmpX Deleted Mutants Of Porcine Extraintestinal Pathogenic Escherichia Coli

Extraintestinal Pathogenic E coli (ExPEC) is one of the most important zoonosis pathogens. It can efficiently colonize the niches outside of the gut and cause diseases via its specific virulence factors. In recent years, many clinical ExPEC strains isolated from pig always show multidrug resistance, which can decrease the effect of antibiotics treatment. Additionally, profound studies on ExPEC pathogenic mechanism have not been performed and high efficient vaccines for the control of ExPEC infection have not been developed. Some ExPEC strains isolated from animal, share the similar virulence factors, population or serotype with the strains isolated from human, which increase the risk of cross infection between humans and animals. In order to control the infection of ExPEC, screening the effective immune protective antigen and developing effective vaccines has the vital implication.OmpX is one of the outer membrane proteins and belongs to the Ail protein family. In other bacteria, OmpX mainly involved in bacterial adhesion, invasion and resistance to the host immune defense, so it is likely to be a candidate targets for vaccine development.In this study, using wild-type strain PCN042isolated from pig lung as parental strain, ompX-deleted mutant was constructed using homologous recombination technique. Then, the biological characteristics including the bacteria antimicrobial susceptibility, biofilm formation and pathogenicity were compared between the WT and mutant, in order to investigate the function of ompX in bacteria. The main results were shown as follows:1. Construction of the△ompX mutant and its complement strain.The upstream and downstream fragments of ompX were amplified from the ExPEC PCN042genome by PCR and linked by overlapping PCR. The linkage product was digested with restriction enzyme and introduced into the suicide plasmid pRE112. The recombination plasmid pRE112-△ompXX/as transferred into E.coli x7213strain. The△ompX mutant was selected by biparental mating between donor strain x7213-pRE112△ompX and recipient strain WT.The full-length ompX gene was inserted into the plasmid pHSG396to acquire the expression of ompX. The recombinant vector was transferred into△ompX strain to select the complement strain.2. Detection of the biological characteristics of the△oompXmutantsThe△ompX mutant can stably inherited when continuous transfer of culture reached 10generations from one tube to another. The growth rate had not significant different between the WT strain and mutants. Determined by using semi-solid plate, the motility of△ompX mutant were significantly stronger compared to WT strain (P<0.01). The biofilm formation detected by microtiter plate assay was no difference between the WT and AompX (P>0.05). In addition, ompX deletion led to the increase in the bacterial sensitivity against tetracycline and cefotaxime.The roles of OmpX in ExPEC virulence were evaluated by in vivo and in vitro experiments. OmpX was essential for bacterial adhesion to and invasion of A549and RAW26.7cell, but didn’t show obvious role in macrophage survival ability. LD50of the△ompX mutant in mice model was6.27times higher than that of the WT strain which verified the essential role of ompX in ExPEC virulence. The infected mice by AompX mutant had significant lower numbers of bacteria load in heart, liver and spleen than the WT strain post-infection8h and24h, and in lung, kidney post-infection24h and8h, respectively (P<0.01). In conclusion, OmpX plays an important role in ExPEC virulence, and is the potential target antigen for ExPEC pathogenesis research and vaccine development.