Type VI secretion system in uropathogenicProteus mirabilis N2

• Main Authors: Yi-Hwa Chou, 周宜樺
• Other Authors: 廖淑貞
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/328vpy

碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 105 === Proteus mirabilis is a frequent pathogens causing urinary tract infection, mainly in patients with the long-term use of urinary catheters. Bacteria have developed diverse regulatory mechanisms for adaptation to the changing environments. Type VI secretion systm (T6SS) is one of the weapons for different bacteria to compete with each other and gain predominance in their niches. It is a protein secretion system which requires cell-cell contact, between bacteria or a bacterium and a eukaryotic cell. Studies have shown that T6SS is regulated by environment-related regulators, but the regulation of T6SS in P. mirabilis remains uncleared. Our transcriptome analysis showed T6SS of P. mirabilis N2 including T6SS main structure gene and four hcp-vgrG effector operons is likely regulated by Crp, Hfq, CpxR, RpoN and RpoE. We observed dienes lines formation between wild-type and crp, hfq, cpxR, rpoN and rpoE mutant strains. The wild-type has growth predomince over all mutant strains and all mutant strains are killed by the wild-type. It means all above regulators and sigma factors affect killing ability of P. mirabilis. We found the P. mirabilis genome contains one T6SS main structure (17 genes) operon and four T6SS hcp-vgrG effector operons by sequence specific reverse transcription PCR. Realtime PCR and promoter reporter assay demonstrated expression of T6SS main structure and/or four hcp-vgrG effector were up-regulated by Crp, Hfq, CpxR, RpoN and RpoE. We further found that Crp directly binds to the promoter regions of T6SS structure and four effector operons by DNase I footprinting analysis. In addition, crp-overexpression strain showed better killing ability against the wild-type. We used bioinformatic tools and found 3 putative toxin genes in T6SS of P. mirabilis N2. Toxin-overexpression strain also exhibited better killing ability than the wild-type and E. coli. Altogether, our study suggests that Crp, Hfq, CpxR, RpoN and RpoE positively regulated T6SS of P. mirabilis N2 and affected its killing ability.