表現鉤端螺旋體Omp52外膜蛋白質的大腸桿菌在巨噬細胞之存活

The survival of E. coli expressing leptospiral outer membrane protein Omp52 within macrophages

陳韋廷(Wei-Ting Chen)

指導教授 ： 劉振軒

共同指導教授 ： 潘銘正(Ming-Jeng Pan)

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01544

摘要

鉤端螺旋體症 (leptospirosis) 是廣泛流行於全球的人畜共通傳染病，是由鉤端螺旋體所造成，鉤端螺旋體症在台灣的主要流行血清型為Leptospira santarosai serovar Shermani。分析病原性L. santarosai serovar Shermani及非病原性L. biflexa serovar Patoc基因體的差異，選殖出23個DNA片段只存在於病原性L. santarosai serovar Shermani而不存在於非病原性L. biflexa serovar Patoc。C67片段經BLASTX程式比對可能為一個外膜蛋白質，所轉譯之蛋白質為 Omp52，有1371 bp可轉譯成456個胺基酸，分子量爲52.6 kD，其C端含有OmpA的Domain，為OmpA familiy外膜蛋白質。在E. coli，必須有OmpA外膜蛋白質表現才能有效地讓大腸桿菌進入人類與鼠類的巨噬細胞裡，並且讓大腸桿菌在巨噬細胞中存活與複製，最後甚至因細菌大量複製而使得巨噬細胞破裂。病原性鉤端螺旋體也會造成巨噬細胞的細胞凋亡（apoptosis），免於被巨噬細胞的清除作用所殺滅而致病，此情形與大腸桿菌有些類似。目前鉤端螺旋體Omp52外膜蛋白質的功能仍是未知，由於其結構含有OmpA的Domain，且只存在於病原性鉤端螺旋體，藉由進行invasion assay了解有鉤端螺旋體Omp52外膜蛋白質表現時，是否可使大腸桿菌於巨噬細胞中存活，以評估Omp52與避免被吞噬作用的清除是否有關聯？以BL21大腸桿菌進行實驗後，結果顯示可表現鉤端螺旋體Omp52外膜蛋白質的實驗組BL21 (pWJN1)在細菌與細胞比例MOI= 0.1、MOI= 1時，於巨噬細胞中的存活量都大於對照組，並達到顯著差異性 ( p < 0.05 )，於MOI= 10時，存活數量達到極顯著差異（p < 0.001)。再以刪除OmpA外膜蛋白質基因的大腸桿菌E98進行實驗，讓E98表現Omp52外膜蛋白質，並讓另一實驗組表現鉤端螺旋體另一外膜蛋白質LipL32，以探討是否有類似於Omp52的功能。在MOI= 1、MOI= 10時，會表現Omp52的E98大腸桿菌存活量比無法表現Omp52的大腸桿菌好，並達到統計學上顯著差異性（p < 0.05）。不論是以BL21或以E98大腸桿菌進行invasion assay，兩者結果均顯示鉤端螺旋體Omp52外膜蛋白質有助於大腸桿菌在巨噬細胞之存活的功能，並且使大腸桿菌於細胞中仍能複製。此外，實驗結果也顯示會表現鉤端螺旋體LipL32外膜蛋白質的E98大腸桿菌於巨噬細胞中的存活量與對照組並無差異，表示LipL32無法幫助大腸桿菌在巨噬細胞的存活。
本論文另一部分是要了解動物感染鉤端螺旋體期間，鉤端螺旋體是否有表現Omp52外膜蛋白質。以免疫化學染色的方法，於L. interrogans serovar Manilae strain菌體上偵測到Omp52外膜蛋白質的表現。但在注射低劑量與高劑量鉤端螺旋體攻毒的倉鼠腎臟組織，使用免疫組織化學染色法與間接免疫螢光法均無法偵測到Omp52的表現，表示此實驗動物感染L. interrogans serovar Manilae strain時，菌體無Omp52之表現。

關鍵字

鉤端螺旋體 ； 外膜蛋白質

並列摘要

Leptospirosis is a worldwide zooanthroponosis casused by bacteria belonging to the Leptospira. In Taiwan, leptospirosis is caused mainly by Leptospira santarosai serovar Shermani. We isolated 23 DNA fragments present in pathogenic Leptopsira santarosai serovar shermani but absent in non-pathogenic Leptospira biflexa serovar patoc. An open reading frame of 1371 bp encoding a protein of 456 amino acids (designated omp52) with a predicted molecular mass of 52.6 kD is matched to the clone C67. Omp52 gene identified among pathogenic L. santarosai serovar Shermani strain CCF but absent in non-pathogenic L. biflexa serovar Patoc. It was shown to be an outer membrane protein containing a C-terminal OmpA consensus domain and exposed on the cell surface. OmpA expression is necessary for effective binding to and subsequent uptake of E. coli by human and murine macrophages, and OmpA+ E. coli can replicate in the phagocytic cells. Infected with OmpA+ E. coli macrophages will disrupt after postinfecion. Pathogenic leptospires survive by evading phagocytosis and induct the apoptosis of macrophages. This phenomenon is similar to OmpA+ E. coli. The function of Omp52 is still unknown. Because there is a OmpA consensus domain on Omp52 structure, and Omp52 only present in pathogenic Leptopsira. We used invasion assays to investigate the interactions of transformed E. coli with macrophage cell lines. The amount of survived bacterium will reveal whether evading phagocytosis is related to Omp52 in pathogenic leptospires. This study is divided into two parts, E. coli BL21 (OmpA+) and E98 (OmpA- E. coli). Using BL21 (pWJN1) which can express leptospiral outer membrane protein Omp52 to perform invasion assays. The results of invasion assay which were carried out with 10-fold less ( MOI=0.1) and MOI of 1 showed BL21 (pWJN1) survival better than E. coli without Omp52 expression ( p < 0.05 ). In 10-fold more ( MOI=0.1), BL21 (pWJN1) also survival better than E. coli without Omp52 expression ( p < 0.001）. E98 （OmpA- E. coli） was transformed with pWJN1 containing the Omp52 gene. In MOI= 1and MOI= 10, the amount of E98（pWJN1）in macrophages are lager than E. coli without Omp52 expression （p < 0.05）. The results reveal one of Omp52 functions is to help evade phagocytosis, and E. coli which express Omp52 can still replicate in macrophage cell lines. Furthermore, E98 was transformed with pRSETc-LipL32 to express the other leptospiral outer membrane protein LipL32. The invasion assays results showed LipL32 can’t help bacterium survival in macrophages.

並列關鍵字

leptospira ； Omp52 ； outer membrane protein

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