Research On The Toxic Effect Of An Avian Acinetobacter Baumannii Strain And Function Of Surface Antigen 1 (SurA1)

Acinetobacter baumannii(A. baumannii, Ab) is a common kind of Gram-negative bacterium, which belongs to the conditioned pathogen and only endangers the immunocompromised group. In recent years, with the gradual increase of hospital-acquired Ab, the harm done by Ab has gradually aroused people’s attention.There is growing evidence that the drug resistance and toxic effect of Ab are being enhanced. According to statistics, the occurrence rate of multi-drug resistant strain of Ab is increasing by 3% every year. The number of death caused by Ab has reached over ten in the United States every year. As research continues, it has been found that the bacterium has strong mutation ability and environmental adaptability, widely distributed in various environments, threatening the human health at any time. Due to the limitation of bacteria identification technology in earlier years, it was unknown that Ab could also cause the animal infection. With the advent of molecular biological technique, the infection incident of Ab can be clearly identified, and it is also found that there are gradually more and more Ab incidents infecting animals. It may be shocking that most Ab strains infecting animals are from human infection strains;therefore, the researchers find that Ab has been zoonotic.In 2011, 3000 chicks were directly died and tens of thousands of chicks were sick in a chicken house in Jilin because the fodder was polluted by Ab, causing severe economic losses. Our team identified that the main pathogen causing the chicks death was Ab through the autopsy, pathology observation, pathogen isolation, strain morphological observation, physiological and biochemical test and gene sequence analysis, and we named the separated strain CCGGD201101. Through the regression experiment, CCGGD201101 strains in the chicks infected for one week and six to eight weeks presented the same lethal infection ability. Ab often colonizes the human or animal skins and does not cause the animal disease, but the extensive chick death indicates that Ab is gradually more pathogenic to animals and zoonotic. The research group has been attracted by the questions that why the conditioned pathogen without pathogenicity to animals originally obtains the corresponding pathogenic ability, andwhat the main pathogenic factor and pathogenic mechanism are.In the preliminary study, we screened out more than 30 proteins whose expression level is up-regulated significantly in CCGGD201101 strain through the fluorescent two-dimensional difference electrophoresis with CCGGD201101 strain as the test strain and ATCC17978 as the reference strain. Through the database search and literature review, it was found that these differentially expressed proteins were closely related to the physiological functions of Ab, and some might play important roles in the toxicity effect and environmental adaptability of Ab.The first experiment in this research mainly proved that CCGGD201101 strains had strong toxic effect and environmental adaptability. Through the infection experiment of chicks, Galleria mellonella and mice, it was proved that CCGGD201101 strains has significantly enhanced toxic effect compared to ATCC17978 and ATCC19606. Then we compared and analyzed the proliferation rate,biological membrane formation, secretion level of iron carrier and biological serum killing resistance, and proved that CCGGD201101 strain had strong environmental adaptability.In the early fluorescent two-dimensional difference electrophoresis and fluorogenic quantitative PCR experiment, it was proved that the expression level of Surface antigen protein(Sur A1) obviously increaseing CCGGD201101 strain significantly, and it was also found in other studies that the up-regulated expression level of this protein was related to the toxic effect and enhanced drug resistance of Ab,so this protein caught our attention. To study the function of Sur A1, we prepared the recombinant Sur A1 protein and monoclonal antibody that could identify the natural Sur A1 protein for the first time. Through comparing the building methods of various gene mutant strains, we chose the gene substitution method to build the Sur A1gene-deleted strains, and prepared the corresponding gene covering strains. We analyzed the toxic effect of the wild-type CCGGD201101 strain, Sur A1 gene-deleted strain and gene covering strain on G.mellonella, also analyzed the influence of Sur A1 on the in vitro competition parameter of strain and within-host invasion efficiency,and then analyzed various environmental adaptation capacities, including the drought resistance, ultraviolet radiation resistance, biological membrane, serum killing resistance and movement ability, and the experimental results proved that Sur A1 played an important role in the toxic effect and environmental adaptability ofCCGGD201101 strain.At present, preventing Ab with the immunization is an important way to restrain the nosocomial infection. Omp A protein is the virulence-associated protein of Ab studied most thoroughly, and it has been proven in our research that Sur A1 protein is closely related to the toxic effect. So we analyze the protection of Omp A, Sur A1 and two recombinant proteins for BALB/c mice to evaluate the application value of these two kinds of proteins as the recombinant protein vaccine candidate protein.Overall, in this research, we proved that CCGGD201101 strain was the Ab strain with enhanced toxic effect and strong environmental adaptability through the animal experiments. The biological function of Sur A1 protein from CCGGD201101 strains was studied, and it was proved that the protein played an important role in the toxic effect and environmental adaptability of CCGGD201101 strain. Through the immune protection research, it was finally proved that both Sur A1 and Omp A proteins were immune protection proteins with certain protective function. We hope that the experiment results can provide some references for further study on the toxic effect and vaccine design of Ab.