應用奈米技術探討克雷白氏肺炎桿菌第三型線毛的黏附與延展特性

Abstract

細菌藉由其表面線毛黏附宿主細胞，因線毛具螺旋彈簧結構的伸縮特性，能降低環境沖刷力的影響，使細菌與宿主保持緊密黏附。克雷白氏肺炎桿菌(Klebsiella pneumoniae)是一株伺機性感染細菌，近年來臨床上發現多種抗藥性菌株，使得治療方式趨於困難，而其表面第三型線毛引起細胞製造生物膜，進而產生抗藥性。本論文將以克雷白氏肺炎桿菌主要致病的因子第三型線毛作為材料，利用雷射鑷夾(OT)、電子顯微鏡（TEM）、原子力顯微鏡（AFM）、基因重組技術、基因標定技術、胺基酸定點突變技術等奈米技術，從生物物理的角度，探討單一根第三型線毛的特性，並進一步分析影響線毛主要蛋白MrkA組裝的重要殘基。我們發現第三型線毛的長度約為1微米，線毛的螺旋截面並不對稱，截面最大寬度7.4奈米和最小寬度4.2奈米。第三型線毛頂端黏附蛋白有四個黏附因子，其中MrkDv3與膠原蛋白的黏附力最大，約為4皮牛頓，是黏附蛋白的重要因子。第三型線毛受力伸展曲線具有三個階段：(1)第一階段表現線性的受力伸長特性，彈性係數為60.9皮牛頓/微米，楊氏模數為100皮牛頓/微米2；(2)第二階段表現固定力伸展特性，野生株的線毛以固定力66 ± 4皮牛頓拆開並伸展數微米長；(3)第三階段表現非線性的受力伸長特性，其受力伸展曲線轉折點為特徵力102 ± 9皮牛頓。另外，針對第三型線毛骨幹主要蛋白MrkA單體，我們篩選十二個MrkA關鍵的胺基酸進行定點突變，以分析其受力伸展物理特性的變化。其中G189A突變株具特殊力學性能，其第二階段的拆解力呈現持續增加的拆解力(57~68皮牛頓)，不同於其他突變株或一般株以固定66皮牛頓力拆解。這樣的結果顯示，G189胺基酸對MrkA的結構拆解力穩定度扮演重要的角色。本論文提供一個研究各種線毛的生物力學性能的奈米技術平台，藉由線毛生物物理特性結果與發現，能提供細菌感染致病的資訊，有助於傳統細菌感染與抗藥性的研究。

Fimbriae exhibiting flexible and stretchable properties similar to springs may aid bacteria in remaining attached to host cells by reducing the impact of the flushes in environment. Klebsiella pneumoniae is a common pathogen causing both pneumonia and urinary tract infections. Most pathogenic Klebsiella pneumoniae strains produce type 3 fimbriae, which are critical for bacterial biofilm formation. In this thesis, we investigated the structural and mechanical properties of Klebsiella pneumoniae type 3 fimbriae, which constitute a known virulence factor for the bacterium, and to identify residues in the major pilin MrkA important for pilus assembly. Type 3 fimbria was 1 micron in length, and the largest diameter was 7.4 nm and the smallest was 4.2 nm, have an elliptically cylindrical structure with the largest diameter and smallest diameter of each ellipse-like cross section in fimbrial structures by using transmission electron microscopy. We present type 3 fimbriae MrkD adhesin variants, mrkDV3-,was the best adhesin, and the adhesive force between mrkDV3-expressed fimbriae and collagen IV is about 4 pN.The force-extension curve of type 3 fimbria exhibited a three-phase：(1) the first spring-like phase stretched with increasing force, the spring constant kp of 60.9 N/μm, the estimated Young’s moduli (E) were 100 Mpa, (2) the second uncoiling phase, it started to uncoil and extended several micrometers at a fixed force of 66 ± 4 pN, and (3) the final nonlinear phase with a characteristic force of 102 ± 9.8 pN at the inflection point. In addition, we screened twelve critical residues on major pilins MrkA of type 3 fimbriae. By contrast, the G189A mutant remained capable of producing normal amounts of fimbriae. Further investigating the mechanical properties of the G189A fimbriae revealed that the uncoiling force for MrkA-MrkA interaction varied from 58 pN to 67 pN rather than be constant like the uncoiling force of 66 pN measured from the other mutants or wild type. The work has provided a method that allows us to understand of the biomechanical function of different types of pili. The investigation of pili brings information for the design of new drugs to prevent bacterial infections, which is increased bacterial resistance towards antibiotics.