尿道致病性奇異變形桿菌KdpDE雙組成調控系統之基因調控暨功能分析研究

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，以及免疫功能低下的病患中造成伺機性感染。鉀離子 (K+) 對於細菌維持生理功能以及細菌毒力與致病力皆占有重要的角色，已有研究顯示，在Salmonella中，當鉀離子transporter缺失，會造成分泌蛋白的減少以及降低對上皮細胞的侵入能力。KdpDE雙組成系統是細菌中很廣泛存在的histidine kinase/response regulator系統，當環境中可利用的K+濃度受到限制時，KdpD會進行自我磷酸化並轉移磷酸根到胞內的KdpE，而磷酸化的KdpE會誘發KdpFABC K+ transporter表現，而當環境中K+濃度變高時，會抑制KdpDE訊息傳遞，抑制KdpFABC的表現，藉此幫助調節胞內K+的濃度。已有許多研究顯示，kdpDE對於細菌致病力是很重要的，例如Yersinia pestis的kdpDE突變株比起野生株在嗜中性球存活率較低。另外在Salmonella Typhimurium中，kdpD突變導致感染線蟲的能力以及在巨噬細胞中的存活率降低，也影響在高鹽以及hydrogen peroxide (H2O2)環境中的存活率。綜觀鉀離子對細菌的重要性，而KdpDE會調控KdpFABC鉀離子攝取系統，因此本文的目的欲探討KdpDE 在P. mirabilis的角色及其調控。 我們搜尋P. mirabilis N2 genome上的kdpFABCDE，發現其胺基酸與E. coli 、S. Typhimurium之相似度皆在60%以上。首先以reporter assay確認了低鉀環境下可以誘發kdpF promoter的活性，高鉀則會抑制。因而以低鉀的環境下分析kdp operon，結果顯示kdpFABCDE為同一個operon。之後構築kdpE突變株，分析野生株、kdpE突變株、kdpE互補株與kdpE過度表現株之表現型，發現突變株與野生株並無差異，然而，過度表現株在表面移行能力、抗酸能力、抗H2O2與抗高鹽能力上，都優於野生株。接著reporter assay顯示野生株在LB時與kdpE突變株同樣不會誘發kdpF promoter的活性而kdpE過度表現株則會誘發kdpF promoter的高活性。於是找尋可以誘發野生株kdpF promoter活性之因子，發現野生株在低鉀、高鈉、H2O2、酸及高糖的環境下皆可活化kdpF 的promoter活性，kdpE突變株則否，顯示在這些壓力下可活化KdpDE訊息傳遞。接著，以高鈉或低鉀活化KdpDE訊息傳遞之環境下分析kdpE突變株與野生株的表現型，結果顯示在抗鹽、抗氧化壓力與抗酸能力上野生株優於突變株。另外，發現kdpE磷酸化位點突變仍然可以活化kdpF promoter，但磷酸化的KdpE比非磷酸化的KdpE調控的能力更強。也觀察到kdpD突變株不管在表現型或是對於kdpFABC operon的調控上，結果皆與kdpE突變株一致，暗示在低鉀或高鉀環境下並不會有別條路徑透過KdpE去調控kdpF promoter。最後我們也探討KdpDE accessory proteins對KdpDE訊息傳遞之影響，發現PtsN與KdpF蛋白質過度表現皆會抑制kdpF promoter活性，且Bacterial-two hybrid assay顯示PtsN與KdpD之間有交互作用。 總之KdpDE會正向調控kdpFABC表現，非磷酸化的KdpE還是可以活化kdpF promoter，低鉀、高鈉、H2O2、酸及高糖都是KdpDE雙組成系統的訊號，低鉀、高鈉可以誘發下游基因保護細菌免於氧化壓力、酸性以及高鹽環境的傷害。

Proteus mirabilis with the swarming characteristic often causes urinary tract infections occurring mainly in patients with the long-term implantation of urinary catheters. According to previous study, potassium plays an important role in maintaining the function, pathogenicity and toxicity of bacteria. The lack of potassium transporters results in the decrease of secretion proteins and the invasion toward epilethial cells of Salmonella. KdpDE two-component system is a wildly expressed kinase/response regulator in bacteria. Intracellular KdpE is phosphorylated by KdpD when encountering a decreased concentration of potassium, which enhances the expression of KdpFABC potassium transporter. On the other hand, the increased concentration of potassium inhibits KdpE signal then decreased the expression level of KdpFABC to regulate the intracellular concentration of potassium. It has been proven that KdpDE is crucial to the pathogenicity of bacteria such as Yersinia pestis kdpDE mutant is with lower survival than wild type, Salmonella Typhimurium kdpDE mutant leads to a lower infectious rate to nematode and lower survival rate in marcophages as well as in high salinity condition. KdpDE regulates the KdpFABC and influences the uptake of potassium. The aim of this research is to investigate the gene regulation and function of the KdpDE two-component system in uropathogenic Proteus mirabilis. We discovered that kdpFABCDE in N2 genome of P. mirabilis is with 60% similarity with E.coli and S. Typhimurium. The kdpF promoter activity showed the same expression pattern as E.coli and S. Typhimurium in response to the potassium concentration. We proved kdpFABCDE can be a transcriptional unit under low potassium. Subsequently the phenotype of kdpE mutant, wild type, kdpE complementation and kdpE overexpression were analyzed. The tolerance to acid, H2O2 and high salt of the kdpE overexpression strain but not mutant was significantly better than the wild type. We showed kdpF promoter activity was almost no expression in both wild type and mutant in LB, instead of being highly induced in the kdpE overexpression strain. We then identified low K+, high Na+, H202, H+, or glucose as signal to trigger expression of kdpFABC through the kdpDE signal transduction pathway. Under high Na+and low K+ concentration, the salt-resistant, acid-resistant and anti-oxidation ability of wild type are significantly better than of mutant. We also demonstrated un phosphorylated KdpE still can induce kdpF expression in a less extent. Besides, kdpD mutant had no difference with kdpE mutant in terms of the phenotypes and the regulation of kdpFABC operon, suggesting kdpF promoter is solely regulated by kdpDE pathway under the condition used low K+, high Na+ condition. Moreover, overexpression of PtsN inhibited the activity of kdpF promoter and bacterial two-hybrid assay showed interaction between KdpD and PtsN. In summary, KdpDE regulated the expression of kdpFABC no matter KdpE is phosphorylated or not. Low K+, high Na+, high H+, and high H2O2 are signals of the KdpDE two component system. Low K+or high Na+ induces expression downstream gene of the KdpDE system to protect bacteria from the damages of H2O2 stresss, high saltanity and highly acidic condition.