Temporal pore formation-mediated egress from macrophages and alveolar epithelial cells by Legionella pneumophila

Legionella pneumophila does not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellul ar replication (L.-Y, Gao and Y. Abu Kwaik, Environ. Microbiol, 2:79-90, 20 00), In contrast to this single mode of killing of protozoa, we have recent ly proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis duri ng early stages of the infection, followed by an independent and temporal i nduction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages by L. pneumophila is mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified fiv e L. pneumophila mutants defective in the pore-forming activity. The phagos omes harboring the mutants do not colocalize with the late endosomal or lys osomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular re plication, the mutants are defective in cytotoxicity and are "trapped" with in and fail to lyse and egress from macrophages and alveolar epithelial cel ls upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic deat h of the host cell. Data derived from cytotoxicity assays, confocal laser s canning microscopy, and electron microscopy confirm the defect in the mutan ts to induce necrosis of macrophages and the failure to egress from the hos t cell. Importantly, the mutants are completely defective in acute lethalit y (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the pore-forming activity of L. pneumophila is not required for phagosomal tra fficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis o f infected macrophages to allow egress of intracellular bacteria. In additi on, this activity plays a major role in pulmonary immunopathology in vivo.