The Pore-forming Toxin of Legionella Pneumophila

描述（由申请人提供）：嗜肺军团菌的细胞内复制最终导致宿主细胞的裂解和细菌的排出，并伴有肺泡间隙的广泛坏死。我们的数据表明，细胞内嗜肺乳杆菌在细胞内复制终止时触发细胞内形成孔毒素/细胞溶素的表达，并且在形成孔毒素中有缺陷的突变体在细胞内复制类似于亲本菌株，但仍然“被困”在宿主细胞内并且不能从宿主细胞中排出。我们已经确定了成孔毒素及其出口所需的辅助因子，两者都由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，而另一种蛋白质是细胞溶解素和其他毒力效应物出口的辅助因子。嗜肺乳杆菌的细胞溶素在肺部病理中起着重要作用，并有可能成为一种有效的军团病疫苗。为了验证这一假设，我们的具体目标是研究以下内容：1)鉴定两种蛋白质的功能结构域和鉴定细菌出口所需的溶胞素相互作用蛋白；2)细胞溶解素孔的插入对细胞生物学的影响，包括吞噬体和细胞器；3)溶细胞素在急性炎症中的作用及其作为疫苗的潜在用途。研究意义：我们提出的研究将在分子、生化和细胞水平上揭示嗜肺乳杆菌在利用宿主细胞增殖后产生细胞毒性、杀死宿主细胞并从宿主细胞中排出的新机制。我们的研究将评估这种毒素作为军团病疫苗的潜在用途。嗜肺乳杆菌的成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），也可用于治疗目的，例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其他空泡细胞内病原体如分枝杆菌、沙门氏菌和衣原体提供一个范例。

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.