克雷白氏肺炎桿菌性腦膜炎：細胞分析模式的建立，致病因子的分離鑑定和致病機制的探討

Abstract

克雷白氏肺炎桿菌屬一伺機性感染的細菌，臨床上常造成菌血症、尿道和呼吸道感染。此菌在台灣糖尿病病人化膿性肝膿瘍、眼內炎、腦膜炎和敗血性肺栓塞的顯著比例，特別受到我們國內醫生、感染專家與學者的重視。最近國內外各大醫院均報導，成人和幼兒罹患克雷白氏肺炎桿菌腦膜炎的病例有日漸增多的趨勢，同時因為克雷白氏肺炎桿菌高比例的抗藥性問題，阻礙了抗生素治療的時效性，而造成病人死亡或嚴重的後遺症，針對新目標做新藥開發的工作更是刻不容緩。我們希望了解克雷白氏肺炎桿菌腦膜炎的致病機轉，藉著干擾這些克雷白氏菌致病相關基因的功能來發展專一而有效的治療藥物或疫苗。我們未來二年的研究規劃如下： （一） 第一年：我們計劃和台北榮總馮長風醫師合作，收集及分析克雷白氏肺炎桿菌腦膜炎菌株。首先進行血清學及各種分子的分型法分析，以了解是否有一特殊菌株與腦膜炎有關；同時，我們將建立細胞分析模式以提供探討這些菌株引起腦膜炎的可能機轉。我們進一步將藉由美國國家生物資訊中心的分析比對軟體，由已公佈克雷白氏肺炎桿菌的基因體核酸序列中，找出和致病相關的目標序列，包括特殊的黏附蛋白、侵入因子等，加上我們實驗室所發現數個與毒性調控相關的候選基因，來設計特定的引子組以聚合脢鏈鎖反應分析所收集的腦膜炎菌株。而對存在於這些腦膜炎分離株且未曾被仔細分析的致病候選基因，我們將進行基因選殖的工作，並藉由大腸桿菌的表現系統來表現這些致病相關的蛋白質。 （二） 第二年：我們將設法大量生成這些致病相關的基因產物，並純化這些重組蛋白，更進一步作生物及生化活性的測試。並藉本實驗室已建立克雷白氏菌的基因突變系統，來作基因剔除實驗，希望比較分析這些突變株和野生株的生物活性，譬如對黏附或入侵細胞株的能力和對小鼠毒性的改變，來了解這些致病相關的基因在其致病機轉中扮演的角色。確定這些基因和細菌致病能力的相關性後，我們將進一步研究調控這些基因表現的因子，研擬可能的標的發展藥物來干擾細菌致病的機制，達到控制和治療克雷白氏菌腦膜炎的目的。

Klebsiella pneumoniae is an important opportunistic pathogen which causes bacteremia, and urinary and respiratory tract infections. The tight association between K. pneumoniae liver abscess, endophthalmitis, and diabetes in Taiwan has especially drawn a lot of concerns. In Taiwan, an increasing number of K. pneumoniae meningitis have also been reported in pediatric wards and adult patients. However, many clinical isolates of K. pneumoniae are highly resistant to antibiotics. The appearance of the extended spectrum beta lactamase producing K. pneumoniae (ESBLKp) further limits the effectiveness of the current therapy. Drug development to specific target is warranted for an antibacterial modification. We plan to study the pathogenic mechanism of K. pneumoniae meningitis and search for a specific drug target or vaccine candidate for a novel antibacterial means. The specific aims to be finished in two years are: The first year: We plan to collaborate with Dr Chang-Phone Fung in Taipei Veteran General Hospital for K. pneumoniae meningitis isolates. We will initially focus on basic bacteriologic studies and molecular epidemiologic studies. We will also establish a cell assay model system to analyze the likely pathogenic mechanism. We will also work on comparative analysis of the contig sequences in PDB to isolate the potential virulence genes and to functional analyze the gene products. The second year: We will generate specific gene-disruption mutants and analyze effects of these mutations for their functional roles on bacterial pathogenicity. To determine the regulatory mechanism for the expression of the genes that may help to explore a pool of potential targets.