- 學位論文
奇異變形桿菌中雙組成訊息傳遞系統調控多黏菌素B的感受性、表現移行及相關致病因子之研究
Characterization of two-component systems regulating polymyxin B susceptibility, swarming and virulence in Proteus mirabilis
摘要
Proteus mirabilis(奇異變形桿菌)屬於腸桿菌科，革蘭氏陰性的兼性厭氧菌，在健康人腸道內為正常菌叢，但在腸道以外的地方則會造成伺機性的感染。最常見的即為泌尿道感染(UTI)，為院內感染的常見菌。P. mirabilis的特徵是當其處於固體表面時，會從單一細胞的vegetative cells變化成多細胞、多鞭毛具有表面移行能力的swarmer cells，由文獻已知P. mirabilis表面移行的能力與其他致病因子有關，例如溶血酶以及尿素酶活性等。 此一菌株早在1972年即被發現對polymyxin B (PB)具有很強的抗藥性，目前已知是因為其LPS(lipopolysaccharide)的lipid A上面的磷酸根被aminoarabinose所修飾，使得外膜的負電荷被中和，而PB就無法利用其正電荷和細菌外膜結合而失去殺菌能力。 在其他革蘭氏陰性菌，如：Escherichia coli、Salmonella Typhimurium等，PB的感受性與LPS的修飾作用有關，而這些修飾作用可藉由雙組成系統所調控。其中Salmonella Typhimurium LPS分子的修飾同時受PhoP/PhoQ、PmrA/PmrB、RcsC/YojN/RcsB三種system所調控。 我論文的目的是想看在P. mirabilis中是否也存在著雙組成調控系統和PB的感受性有關。由於PB的感受性和swarming都和雙組成調控系統及菌體envelope構造有關，故同時觀察這些雙組成調控系統和swarming及其他致病因子間的關係。首先我們在已發表的P. mirabilis基因體中尋找和Salmonella PhoP/PhoQ相似的蛋白質及基因序列，再將實驗室菌株P. mirabilis P19的目標基因定序出來，一方面以生物資訊學做分析，一方面挑選突變菌株以作進一步的研究。目前找到的兩個雙組成調控系統，其中一個(RppA/RppB, Regulator of Polymyxin B susceptibility in Proteus)和PB的抗藥性有關，突變菌株的感受性較野生菌株上升大於1000倍。我們也發現到當PB不存在時，rppA基因的突變會造成表面移行、LPS pattern、鞭毛蛋白以及溶血酶活性的改變。此外，當PB不存在時，野生菌株及rppA突變株的外膜組成幾乎相同；而當PB存在時，野生菌株可感知此訊息而產生一新的蛋白質。不同於外膜的變化，rppA突變株的LPS不管在有無PB存在時皆和野生菌株不相同，此外也發現PB可以刺激野生菌株的LPS產生更多的變化，而rppA突變株中則沒有此種情形。因此推測這些改變可能皆與PB的抗藥性有關，而RppA/RppB系統的調控參與在其中。我們也觀察到在PB的刺激下，野生菌株的許多致病因子(表面移行、溶血酶、生物膜生成等)會受影響而產生不同於rppA突變株的變化，推測PB可能為此雙組成調控系統的訊息因子之ㄧ。P. mirabilis中有雙組成系統可調控其對PB的感受性、表面移行及致病因子的現象是在本實驗室首次發現。而用Real-Time PCR的方法也發現在PB以及低鎂離子的環境中，rppA基因的表現量有上升，這和過去Salmonella的PhoP/PhoQ系統有正向自我調控的現象相符合。而另一雙組成調控系統(RhpA/RhpB, Regulator of haemolysin in Proteus)和PB的關係不明顯，主要可能和溶血酶以及入侵細胞能力的調控有關係。
並列摘要
Proteus mirabilis is a facultative Gram-negative bacterium and a member of the family Enterobacteriaceae. It is an opportunistic pathogen frequently causes urinary tract infection (UTI) in hospital patients. One of the characteristics of P. mirabilis is its dramatic morphological change on solid surface. When growing on solid surface, it changes from single rod-shaped swimmer cell to an elongated multicellular swarmer cell and it can swarm on agar plate resulting in a bulled-eye pattern. P. mirabilis produces many virulence factors such as haemolysin and urease whose production is co-ordinated with swarmer cell differention. P. mirabilis is naturally resistant to PB, which is a kind of cationic antimicrobial peptides. It is known that the modification of the lipid A of P. mirabilis by aminoarabinose results in PB resistance. In E. coli and Salmonella Typhimurium, LPS modification regulated by two-component system causes the change of PB susceptibility. We reported here that there is a two-component system (we call RppA/RppB, Regulator of Polymyxin B susceptibility in Proteus) which is associated with PB resistance, swarming and virulence in P. mirabilis. The PB susceptibility elevates more than 1,000 folds in rppA mutant than in wild-type. In the absence of PB, we observed rppA mutation results in the change of haemolysin activity, swarming, LPS pattern and flagellin production.We demonstrated PB caused significant change in LPS and OMP profiles in wild-type but not in the mutant. This imply that both LPS and OMP change modulated by RppA pathway involve in the resistance of PB in P. mirabilis. Furthermore, we find that PB has effect on haemolysin activity and biofilm production through the RppA/RppB pathway. To our knowledge, this is the first finding that a two-component system regulates PB resisitance, swarming and virulence in P. mirabilis. In addition to RppA/RppB, we also found another two-component system that has high identidy to Salmonella PhoP/PhoQ in P. mirabilis. The system has no obvious relationship with polymyxin B susceptibility but play a role in the production of haemolysin and ability of invasion.