- 學位論文
探討尿道致病奇異變形桿菌ptsN(EIIANtr)所扮演的角色及其機制
The roles of ptsN (EIIANtr) and underlying mechanisms in uropathogenic Proteus mirabilis
摘要
奇異變形桿菌(Proteus mirabilis)屬於腸內菌科為健康人類腸道中的正常菌叢，常見為伺機性感染於尿導管植入之病患，是尿路感染的重要病原菌。細菌體內具有多種攝取環境中醣類的運輸系統，其一為phosphoenolpyruvate:sugar phosphotransferase system (PTS)，此運輸系統由Enzyme I ( EI, encoded by ptsI )將磷酸烯醇式丙酮酸 ( phosphoenolpyruvate, PEP )上之一磷酸根 (phosphate, PO43-)轉移至HPr ( encoded by ptsH)，而後再由HPr將該磷酸根轉移至不同的醣類特異性磷酸根攜帶酵素II ( phosphoryl carrier enzymes II, EII，包括EIIA,B,C )上，可攝取醣類同時將磷酸根由EIIB轉移至其上，最終磷酸化的醣類 (phosphoryl carbohydrate)進入代謝系統。過去已有研究指出PTS除了運輸醣類的功能外，尚扮演調控的腳色。除了carbohydrate PTS外，許多葛蘭氏陰性菌尚具有第二種PTS，被稱為氮相關PTS ( nitrogen-related PTS, PTSNtr)。PTSNtr 由EINtr (encoded by ptsP)，NPr (encoded by ptsO)以及EIIANtr (encoded by ptsN)組成，其中ptsO與ptsN坐落於rpoN (54) 操縱子內。目前已有文獻指出ptsNNtr 參與許多調控機制。同時，EIIANtr在不同的磷酸化狀態下似乎具有不同之調控功能，而在棕色固氮菌中，以發現未磷酸化之EIIANtr可影響其細胞壁物質之合成。ptsN已於多種細菌被研究，諸如E. coli、K. pneumoniae，但尚未於Proteus mirabilis探討其調控角色。Mobley 及其團隊曾發現glutamine可增強P. mirabilis HI4320的表面移行能力；而Lee與其團隊中提出可藉由L-glutamine (L-gln)及-ketoglutarate (KG)影響大腸桿菌MG1655的EINtr而增加及減少去磷酸化 (dephosphorylated) EIIANtr之比例，並暗示PTSNtr可感應氮源可利用度。 本研究中，透過建構ptsN大量表現菌株，以觀察ptsN影響之表現型，並建構ptsN磷酸化位點突變大量表現菌株(ptsN(H72A))探討其中磷酸化狀態扮演之角色。結果顯示ptsN大量表現株在LB Lennox agar之表面移行能力較vector control顯著為低，而ptsN(H72A)大量表現株則恢復而與vector control 無差異；藉由添加L-gln與KG來觀察ptsN大量表現株減少之表面移行能力時是否藉由ptsN不同之磷酸化狀態來調控，發現不論添加與否，各菌株之間之表面移行能力並未出現新的變化，暗示此二物質在此條件下不能改變ptsN之磷酸化狀態，但野生株於L-gln中之rpoN表現降低。ptsN大量表現株之尿素酶(urease)活性較vector control顯著為低。ptsN大量表現株對於人類腎臟上皮細胞癌細胞 (A498)與移形上皮細胞癌(NTUB1)之附著力均分別較對照之野生株vector control略為提高；在移動相關基因之調控，qPCR結果顯示野生株於1.5%LB固態培養基時將顯著增加ptsNNtr表現；ptsN 大量表現株中glnA表現量明顯下降，暗示可能藉由抑制細胞內glutamine合成造成磷酸化EIIANtr增加進而影響表面移行能力。ptsN大量表現株與ptsN(H72A)大量表現株分別對於氧化壓力(30mM H2O2)耐受性均較vector control為高；在抗氧化壓力相關基因之調控中，30mM H2O2 環境下之野生型的ptsN表現增加，而 ptsN大量表現株中，sodB、soxR及rpoS表現增加，表示其可能藉由增加抗氧化相關基因表現而能提升對抗氧化壓力之能力。ptsN大量表現株與ptsN(H72A)大量表現株於含有5% NaCl之LB 液體培養基之耐受試驗均較vector control略為提高；在滲透壓相關基因之調控中，5% NaCl環境下並不影響ptsN基因表現，猜測是否藉由EIIANtr與KdpD之蛋白間作用 (protein-protein interaction, PPI)進而活化kdpFABC表現而影響細胞內離子濃度平衡。
並列摘要
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.