The Pore-forming Toxin of Legionella Pneumophila

描述(由申请人提供)：嗜肺军团菌在细胞内的复制最终导致宿主细胞的溶解和细菌的外泄，并伴随着肺泡腔的广泛坏死。我们的数据表明，胞内致孔毒素/溶细胞素的表达在细胞内复制终止后被触发，而致孔毒素缺陷的突变株在细胞内复制类似于亲本菌株，但仍被困在宿主细胞内，无法从宿主细胞中出来。我们已经确定了致孔毒素和它输出所需的辅助因子，两者都由一个双顺反子操纵子编码。我们的假设是：其中一种蛋白质是构成“出口孔”的结构毒素，而另一种蛋白质则是细胞溶血素和其他毒力效应物输出的辅助因子。嗜肺乳杆菌溶细胞素在肺部病理中起重要作用，有可能成为一种有效的军团菌病疫苗。为了验证这一假设，我们的具体目标是研究以下内容：1)鉴定蛋白质中的功能结构域，并鉴定细菌出口所需的溶细胞素相互作用蛋白；2)溶细胞素孔的插入对细胞生物学的影响，包括吞噬小体和细胞质细胞器；以及3)溶细胞素在急性炎症中的作用，以及它作为疫苗的潜在用途。拟议工作的意义：我们拟议的研究将在分子、生化和细胞水平上揭示一种新的机制，即嗜肺性乳杆菌在利用其进行增殖后，成为细胞毒力，杀死并从废弃的宿主细胞中排出。我们的研究将评估这种毒素作为退伍军人病疫苗的潜在用途。嗜肺乳杆菌的致孔毒素可能成为各种生物学研究的工具(类似于许多其他细菌毒素)，也可能用于治疗目的，如其潜在的靶向恶性细胞。我们对嗜肺乳杆菌的研究可能为分枝杆菌、沙门氏菌和衣原体等其他空泡内病原体提供一个范例。

DESCRIPTION (provided by applicant): Intracellular replication of Legionella pneumophila is culminated in lysis of the host cell and bacterial egress, associated with extensive necrosis in the alveolar spaces. Our data show that expression of the pore-forming toxin/cytolysin by intracellular L. pneumophila is triggered upon termination of intracellular replication, and mutants defective in the pore-forming toxin replicate intracellularly similar to the parental strain but remain "trapped" within and fail to egress from the host cells. We have identified the pore-forming toxin and a co-factor required for its export, both encoded by a bi-cistronic operon. Our hypothesis is: One of the proteins is the structural toxin constituting the "egress pore" while the other is a cofactor for export of the cytolysin as well as other virulence effectors. The cytolysin of L. pneumophila plays a major role in pulmonary pathology, and has the potential of an effective vaccine against Legionnaires" disease. To test this hypothesis, our Specific Aims are to study the following: 1) Identification of functional domains in both proteins and identification cytolysin-interacting proteins required for bacterial egress; 2) Effects of insertion of the cytolysin pore on cell biology, including the phagosome and cytoplasmic organelles; and 3) Role of the cytolysin in acute inflammation, and its potential use as a vaccine. Significance of the proposed work: Our proposed studies will uncover at the molecular, biochemical, and cellular levels of a novel mechanism by which L. pneumophila becomes cytotoxic, kills and egresses from the spent host cell after its exploitation for proliferation. Our studies will evaluate the potential use of the toxin as a vaccine against Legionnaires' disease. The pore-forming toxin of L. pneumophila may become a tool for various biological studies (similar to many other bacterial toxins) and also for therapeutic purposes such as its potential targeting to malignant cells. Our studies on L. pneumophila may provide a paradigm for other vacuolar intracellular pathogens such as Mycobacterium, Salmonella, and Chlamydia.