應用雷射鑷夾於克雷白氏肺炎桿菌與膠原蛋白間黏附力之研究

Abstract

細菌黏附到宿主細胞表面是傳染病傳染過程中的第一步驟，因此，比較細菌對宿主細胞的黏附力，是研究黏附作用的必要工作，此一成果將來是用來研發有效的治療藥物或疫苗的重要基礎。為此，本論文希望發展雷射鑷夾成為一種量測黏附力的有效工具。近十餘年來，雷射鑷夾的梯度力日益廣泛被用來捕捉、搬運與操縱單一微小生物體。我們利用傳統水流黏滯力法，校正出雷射鑷夾最大捕捉力和雷射光功率的關係，然後根據恰好能分離細菌與宿主的雷射光功率，可推算所對應的雷射鑷夾最大捕捉力，在力平衡時，雷射鑷夾最大捕捉力即為細菌與宿主之間的黏附力。 本實驗我們使用第五型膠原蛋白代替宿主細胞，再利用雷射鑷夾，分別量測並比較克雷白氏肺炎桿菌珠、移除功能性mrk A纖毛蛋白的突變株、以及還原株（將功能性mrk A纖毛植回突變株）對膠原蛋白的黏附力，比較黏附力強弱。實驗結果顯示，黏附力的強弱依次為：「還原株」、「正常株」、「突變株」，不過其中「還原株」與「正常株」的黏附力接近。雷射鑷夾量測的實驗結果，與傳統塗盤數落菌數的實驗結果趨勢吻合，這證實了「功能性mrk A纖毛」，確實是克雷白氏肺炎桿菌黏附人體的主要介質。

Adherence to host cells by a bacterial pathogen is a critical step for establishment of infection. In order to study the mechanism of adherence, it is necessary to measure the biological force due to adherence. Consequently, optical tweezers become an effective tool for this purpose. In the past decade, the gradient force of optical tweezers has been widely applied in the manipulation of a single small bio object. In this thesis, we use an optical tweezers system to separate a single Klebsiella Pneumoniae, the bacterium, from collagen, the host. We first increase the laser power of the optical tweezers until the bacterium is detached from the collagen coated on the surface of a polystyrene bead. Then, we obtain the magnitude of the adhesive force between the Klebsiella Pneumoniae and the collagen, which is equal to that of the corresponding trapping force provide by the optical tweezers at that specific laser power. Note that we calibrate the relationship between the trapping force of the optical tweezers and the power of the laser by using the water-drag-force method. In this experiment, we use Klebsiella Pneumoniae CG43-S3, Klebsiella Pneumoniae CG43-S3-mrk A, and Klebsiella Pneumoniae CG43-S3-mrk A[pYJ01] as the bacterial pathogen, separately. We found that the adhesive force between Klebsiella Pneumoniae CG43-S3 and collagen is comparable with that between Klebsiella Pneumoniae CG43-S3-mrk A[pYJ01] and collagen. And these two forces are apparently stronger than the adhesive force between Klebsiella Pneumoniae CG43-S3-mrk A and collagen. This result by using optical tweezers agrees with that by using the traditional biology method of plating. Our work proves that the functional mrk A cilium was a key factor that enhances the adherence of Klebsiella Pneumoniae to human body