Characterization of Acinetobacter baumannii A1S_0699 protein and its promoter

• Main Authors: Chih-Wei Yu, 游智暐
• Other Authors: 楊秋英
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/kra8sr

碩士 === 國立中興大學 === 分子生物學研究所 === 106 === Acinetobacter baumannii is an opportunistic gram-negative pathogen that can adapt to a variety of environments. It causes severe nosocomial infections including pneumonia, meningitis, skin soft tissue infections and bacteremia in immunocompromised patients. The emergence of multidrug-resistant strains makes the treatment of A. baumannii extremely difficult. In addition to search for new drugs, vaccination is the most plausible approach to prevent infectious diseases. A1S_0699 (Pase2) is a vaccine candidate previously identified in our laboratory. This protein is not detected in cultured A. buamannii using antibody raised against the recombinant Pase2 while immunized mice were protected from A. buamannii infection suggesting that the expression of Pase2 is regulated. In this study I constructed plasmids carrying DNA fragments containing the complete coding sequence of Pase2 with varied length of the upstream sequences and transformed into A. baumannii. Pase2 protein was detected in A. baumannii harvesting a plasmid with 60- to 200- bp upstream sequences; the highest expression was observed in plasmid with upstream 60- bp. However, Pase2 was barely detectable in plasmid with 300- bp upstream sequence. After demonstrating the specific DNA-protein interaction using the upstream 300-bp DNA fragment, I performed DNA pull down experiments with the upstream 60-bp and 300-bp DNA fragments, respectively. SDS-PAGE showed that the proteins bound to these two DNA fragment are slightly different. Interestingly, I also detected Pase2 transcript in the log phase of the wild type A. baumannii but not that from bacteria at stationary phase. Together, these results suggested that the expression of Pase2 protein in A. baumannii is likely subjected to transcription and post-translational regulation. Next, I examined the phenotypic differences between a Pase2 expressing A. baumannii strain with a mock strain. The results showed that A. baumannii expressing Pase2 protein is more resistant to H2O2 and more virulent in the zebrafish embryos model. Together, these data indicate that Pase2 may protect A. baumannii against the host innate immunity by increasing the resistance to oxidative stress thus enhancing its infectivity.