探討ptsN於尿道致病性奇異變形桿菌中調節毒力因子表達所扮演之角色

Abstract

Proteus mirabilis為革蘭氏陰性兼性厭氧菌，為重要的泌尿道病原菌，主要在長期植入尿導管的病人及免疫功能低下的病患中造成伺機性感染。在許多細菌中，具有磷酸烯醇丙酮酸(phosphoenolpyruvate,PEP)依賴性碳水化合物磷酸轉移酶系統(碳水化合物PTS)，可以催化細菌中許多醣類的攝取並轉化為磷酸化形式。該系統由EI將磷酸烯醇式丙酮酸的磷酸根轉移至HPr，再由HPr將磷酸根轉移到不同醣類的特異性磷酸根攜帶酵素(EII)上，而除了運輸醣類的功能外，碳水化合物PTS還具有多種調節功能。除了醣類PTS外，有些細菌具有另外一種氮PTS (PTSNtr)，該系統由EI Ntr (encoded by ptsP)、NPr (encoded by ptsO)及EIIA Ntr (encoded by ptsN)組成，它們分別是醣類PTS的EI、HPr和EIIA之同源物。目前PTSNtr已被發現跟維持細胞完整性、膜壓力或調控K+攝取等功能相關，且其調控作用與EIIA Ntr磷酸化狀態有關。因此，本研究想探討在P. mirabilis中，EIIA Ntr所扮演的角色及其有無磷酸化造成的影響。 首先，我們在LB的環境分析野生株、ptsN突變株、ptsN互補株與ptsN磷酸化位點突變株之表現型，發現ptsN突變株及ptsN磷酸化位點突變株在表面移行能力、尿素酶活性較野生株差，然而，在抗酸能力、抗H2O2、抗高鹽能力及巨噬細胞內存活能力上，都優於野生株。Bacterial-two hybrid assay顯示不管EIIA Ntr磷酸化狀態為何皆可和senor PhoR有交互作用，接著透過reporter assay發現ptsN突變株及ptsN磷酸化位點突變株在低磷酸鹽的環境下會誘發PhoRB的活性，推測磷酸化的EIIA Ntr可能會抑制PhoRB的訊息傳遞。接著，在低磷酸鹽的環境下分析野生株、ptsN突變株、ptsN互補株與ptsN磷酸化位點突變株的表現型，結果顯示ptsN突變株與ptsN磷酸化位點突變株的抗酸、抗鹽及抗氧化壓力的能力較野生株高。接著建構phoB及phoR的突變株，發現兩突變株的抗酸能力都較野生株差，暗示磷酸化的EIIANtr可能透過抑制PhoRB的訊息傳遞來影響抗酸能力。我們進一步發現在高磷酸鹽的環境下ptsN mRNA及 EIIANtr蛋白質量皆較高，故利用IntaRNA預測可能調控ptsN的small RNA，進而找到gcvB，並發現gcvB過度表達時會降低ptsN的表現量，也在低磷環境發現gcvB表現量會升高，並且觀察到在低磷環境下若gcvB突變則抗酸能力下降，表示GcvB在低磷的環境下可能會經由抑制ptsN的表現量而影響抗酸能力。此外，也觀察到ptsN突變株會使crp mRNA下降而rpoS及spf mRNA量上升，透過Crp-dependent GFP螢光試驗也發現ptsN 突變株會顯著減少Crp之活化態。最後，我們也發現ptsN突變株及ptsN磷酸化位點突變株在低鉀環境下會使KdpDE活性增加，且不管EIIA Ntr磷酸化狀態為何皆可和sensor KdpD有交互作用。 總之，磷酸化的EIIANtr會調控P. mirabilis的表面移行能力及尿素酶活性，也會影響抗酸、抗氧化及抗鹽等能力，其可能是受GcvB調控而在低磷環境透過PhoRB 雙組成系統而影響抗酸能力; 或參與在Crp-Spf-RpoS的調控路徑而影響抗酸能力；也可能是在低鉀環境經由KdpDE雙組成系統去影響抗酸、抗氧化及抗鹽等能力。這是首次在P. mirabilis發現EIIANtr及其磷酸化的狀態與致病性具有關聯性。

Proteus mirabilis with the swarming characteristic often causes urinary tract infections occurring mainly in patients with the long-term implantation of urinary catheters. In many bacteria, it has a phosphoenolpyruvate (PEP)-dependent nitrogen phosphotransferase system (PTSNtr), the system consists of EINtr (encoded by ptsP), NPr (encoded by ptsO) and EIIANtr (encoded by ptsN). At present, PTSNtr has been found to be related to cell integrity, membrane pressure or potassium uptake, and its regulation is related to the phosphorylated status of EIIANtr. Therefore, this study will explore the role of EIIANtr in P. mirabilis and the effects of its phosphorylation. First, we analyzed the phenotypes of wild type, ptsN mutants, ptsN complementaion and ptsN(H72A) in LB, and found that ptsN mutants and ptsN(H72A) decreased the ability of swarming and urease activity. However, ptsN mutants and ptsN(H72A) are superior to wild type in the tolerance of acid, H2O2, high salt, and macrophage. Bacterial-two hybrid assay showed EIIANtr can interact with senor PhoR regardless of its phosphorylated status. Then, we found that ptsN mutant and ptsN(H72A) induce PhoRB activity in the low-phosphorus environment through the reporter assay. Thus, we speculated that phosphorylated-EIIANtr may inhibit the signal transduction of PhoRB. Next, we analyzed the phenotypes of wild type, ptsN mutants, ptsN complementation and ptsN(H72A) in the low-phosphorus environment and discovered that ptsN mutant and ptsN(H72A) are superior to wild type in the tolerance of acid, H2O2, and high salt. Then, we constructed the phoB and phoR mutant and found that the tolerance of acid of both mutants were worse than wild type, suggesting that phosphorylated-EIIANtr may affect the tolerance of acid by inhibiting PhoRB signaling. We further found that the level of ptsN mRNA and EIIANtr protein are both higher in the high-phosphorus environment. Therefore, we predict small RNA which may regulate ptsN by IntaRNA and found gcvB. Next, we found that gcvB overexpression reduced the expression of ptsN, and the level of gcvB mRNA increased in the low-phosphorus environment. Moreover, gcvB mutant decreased the tolerance of acid in the low-phosphorus environment, indicating that GcvB may affect the tolerance of acid by inhibiting the expression of ptsN in the low-phosphorus environment. In addition, we observed that ptsN mutant reduced the crp mRNA and increased the level of rpoS and spf mRNA. Through the Crp-dependent GFP fluorescence assay, we also found that ptsN mutant significantly reduced the activated state of Crp. In addition, we also found that ptsN mutant and ptsN(H72A) increased KdpDE activity in the low potassium environment, and EIIANtr can interact with sensor KdpD regardless of its phosphorylated status. In summary, phosphorylated-EIIANtr can regulate the motility and urease activity of P. mirabilis, and also affect the tolerance of acid, H2O2, and high salt. It may be influenced by GcvB in the low-phosphorus environment and through PhoRB TCS or involved in the regulatory pathway of Crp-Spf-RpoS to affect the tolerance of acid; it may also affect the tolerance of acid, H2O2, and high salt through KdpDE two-component system in the low- potassium environment. This is the first time P. mirabilis has discovered that EIIANtr and its phosphorylated state are associated with pathogenicity.