The roles of ptsN (EIIANtr) and underlying mechanisms in uropathogenic Proteus mirabilis

• Main Authors: JIAN-FENG XIE, 謝建峰
• Other Authors: 廖淑貞
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/dhwxsg

碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 106 === Proteus mirabilis belongs to Enterobacteriae as normal flora in health human and often diagnosed as an oppertunistic infection in patients with urinary-catheter implanted. The phosphoenolpyruvate:carbohydrate PTS, or sugar PTS, which transports and phosphorylates a carbohydrate into the cell also regulate the activities of a vast number of genes, and the parallel transfer cascade nitrogen PTS (PTSNtr), which does not transport carbohydrates but exerts many regulatory functions. The phosphate from phosphoenolpyruvate (PEP) was transferred through two general general phosphotransferase proteins: enzyme I (EI, encoded by ptsI or EINtr, encoded by ptsP in PTSNtr) and histidine protein (HPr, encoded by ptsH or NPr, encoded by ptsO in PTSNtr). In the sugar PTS, HPr subsequently phosphorylates the membrane-bound and substrate-specific transport protein enzyme II (EII) allowing uptake of the sugar. In PTSNtr, NPr phosphorylates EIIANtr(encoded by ptsN), which is not active in transport as the required domains are lacking. ptsO and ptsN were found located in the operon of rpoN (54). The phosphorylation state of EIIANtr may play distinct roles in various conditions as found the dephosphorylated form of EIIANtr affects the synthesis of cyst wall materials in A. vinelandii. The roles of ptsN has not been reported in Proteus mirabilis. Mobley et al suggested that the addition of L-glutamine(L-gln) may strengthen the swarming motility in P. mirabilis HI4320. Lee et al claimed that L-gln and-ketoglutarate (KG) in E. coli MG1655 affect the phosphorylation state of EIIANtr. In this study, we investigated the phenotypes associated with ptsN and the role of phosphorylation states of ptsN by construction of overexpression of ptsN and non-phosphorylated EIIANtr (ptsN(H72A)) strains. The result showed that the overexpression of ptsN but not ptsN(H72A) delayed initiation time of swarming. -KG and L-gln were used to study whether the defect in swarming in the overexpression of ptsN was affected by the phosphorylation state of EIIANtr and the result showed that the compounds may not change the phosphorylation state of EIIANtr in this case but L-gln suppresses the mRNA expression level of rpoN in wild type. The overexpression of ptsN leads to lower urease activity and slighty increased the ability of cell adhesion. In the study of the regulation of motility-associated genes, the expression of ptsN significantly increased on solid surface and ptsN overexpression suppresed the expression of glnA which suggests that the suppression of intracellular glutamine concentration may reduce swarming motility by increasing the phosphorylated EIIANtr. The overexpression of ptsN and ptsN(H72A) increased the resistance of oxidative stress and high salt enviroment which may suggest that the interaction between EIIANtr and KdpD affects the homeostasis of intra-ion concentration by kdpFABC while the expression of ptsN elevated under 30mM H2O2. The expressoin of sodB, soxR and rpoS, which are associated with stress response, increased in the the overexpression of ptsN implied that ptsN improved the resistance under oxidative stress by upregulation the associated genes.