彎曲桿菌可在動物引起腹瀉，同時也是在人類造成腸胃炎的主要細菌之一，近年來才逐漸受重視，因此探討彎曲桿菌的特性及其致病因子更顯現其重要性。而細胞致死腫脹毒素(cytolethal distending toxin, Cdt)在空腸彎曲桿菌的致病上被認為扮演一重要角色，我們的初步分析結果顯示Cdt 對於空腸彎曲桿菌來說應該是一必需的毒素，我們也利用重組CdtA, B, C 三個毒素次單位觀察其對細胞的致病機制，發現重組Cdt 具有抑制細胞週期的功能。因此，本計畫第一年我們將持續研究台灣不同地區分離之空腸彎曲桿菌細胞致死腫脹毒素基因及探討三個毒素次單位對細胞致病的作用機制。細胞脂質筏(lipid rafts)對於微生物的感染或是毒素的內噬及運送非常重要，我們推測彎曲桿菌與其Cdt 極有可能藉由脂質筏來感染宿主細胞，因此第二年期間我們將利用已純化的重組Cdt 蛋白質研究Cdt 次單位與免疫細胞脂質筏交互作用、並利用小鼠初級T cells 探討Cdt 毒素的作用、及探討彎曲桿菌感染細胞引起之發炎反應是否受脂質筏的影響。本計畫第三年將進一步利用超高速離心及蛋白質體學的分析方法找尋脂質筏中CdtA, C 結合的分子或接受體並利用共軛焦顯微鏡研究CdtB 於細胞內的輸送途徑。這些研究結果將有助於了解彎曲桿菌及其細胞腫脹毒素的致病機轉並提供臨床醫師治療彎曲桿菌導致腸胃炎之參考。
Campylobacter sp. are the most common causes of human gastroenteritis as well as livestock diarrhea worldwide. While Campylobacter infections are quit common and often severe, relatively little is known about mechanisms of their pathogenesis. It is thought that bacterial virulence factors are more important for those infectious diseases. Numerous cytotoxins in Campylobacter jejuni have been described, but only cytolethal distending toxin (Cdt) has been well characterized. While the molecular characterization of cdt gene in clinical isolates in Taiwan was still absence. Our preliminary data showed that cdt gene was existed in all clinical isolates, indicated that cdt was essential for clinical diseases. In addition, we also found that purified recombinant CdtA, B, C fusion proteins have their cytopathic effects in the induction of cell cycle arrest. In the first year of this research plan, we will continue analysis of the epidemiology of cdt gene and Campylobacter isolates from different medical centers in Taiwan. We will also examine the molecular mechanisms of CdtA, B, C subunits on epithelial cells. Lipid rafts are crucial for microbe infection and toxins delivery. We hypothesized that Campylobacterand Cdt-mediated immunotoxicity is raft-dependent. In the second year of this plan, the association of three Cdt subunits with lipid rafts on immune cells will be investigated. We will also examine the interaction of Cdt and mouse primary T cells. Whether NF-κB and AP-1 signaling pathway are involved in Cdt-mediated cytopathic effects via lipid rafts will be also determined in our second year of this project. In the third year of this plan, we will investigate the common receptors of CdtA and CdtC by sucrose gradient ultracentrifuge and proteomics approaches. The trafficking pathway of CdtB in toxin-treated cells will be also revealed by Confocal microscopy. These results will help us to understand the complicated processes of Cdt-induced cell pathogenesis and would be beneficial for the clinical diagnosis and treatment of Campylobacter-induced gastroenteritis.
