Temporal Pore Formation-Mediated Egress from Macrophages and Alveolar Epithelial Cells byLegionella pneumophila

Abstract

. In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction 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 five L. pneumophila mutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are "trapped" within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the poreforming activity of L. pneumophila is not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo.

Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila , plaB , Exhibiting Hemolytic Activity

“…Other bacterial factors involved in the destruction of eukaryotic cells, such as the RtxA toxin (12) or the icm/dot gene products (1,36,40), have already been characterized. However, especially in the case of the icm/dot genes, which code for a type IV protein secretion system (52,58), it is possible that these determinants do not represent the structural genes actually accounting for the cell-lysing agent.…”

“…The L. pneumophila RTX toxin RtxA has been shown to contribute to cytotoxicity, to pore formation, and to the initial steps of infection, namely, adherence and entry of the bacterium into host cells (12,13). Pore formation is furthermore an indispensable step in bacterial egress after bacterial intracellular multiplication in both amoebae and macrophages (1,40). Pore formation in L. pneumophila depends on the integrity of several genes of the icm/dot locus (e.g., dotA, icmQ, icmR, and icmT), responsible for the expression of a type IV protein secretion machinery involved in pathogenesis (14,36,40).…”

Comparative assessment of virulence traits in Legionella spp.

“…Biphasic killing of mammalian cells by L. pneumophila (Alli et al, 2000;Gao & Abu Kwaik, 1999b) has been proposed in which apoptosis is first initiated, followed by a temporal induction of necrosis and lysis of the host upon growth transition into the postexponential phase (Alli et al, 2000;Byrne & Swanson, 1998;Kirby et al, 1998). The pore-forming activity mediates lysis of the host cell, and mutants defective in pore-forming activity are defective in lysis of the host cell and are delayed in subsequent egress from mammalian (Alli et al, 2000) and protozoan cells (Gao & Abu Kwaik, 2000b). The C-terminus of IcmT has been shown to be essential for pore-formationmediated bacterial egress from the host cell (Molmeret et al, 2002a, b).…”

“…The C-terminus of IcmT has been shown to be essential for pore-formationmediated bacterial egress from the host cell (Molmeret et al, 2002a, b). Importantly, pore-forming activity plays a major role in pulmonary cytotoxicity and inflammation in experimental animals (Alli et al, 2000;Molmeret et al, 2002b). These pathogenic mechanisms have been studied for L. pneumophila but little is known about their role in the pathogenesis of other species of legionellae.…”

“…The dot/icm loci are essential for enhancement of phagocytosis by human-derived cells (Hilbi et al, 2001), macropinocytic uptake by A/J mice-derived macrophages (Watarai et al, 2001), evasion of lysosomal fusion and intracellular replication (Segal et al, 1998;Vogel et al, 1998), induction of apoptosis (Zink et al, 2002), and pore-formationmediated lysis of the host cell and bacterial egress upon termination of intracellular replication (Alli et al, 2000;Molmeret et al, 2002b). Biphasic killing of mammalian cells by L. pneumophila (Alli et al, 2000;Gao & Abu Kwaik, 1999b) has been proposed in which apoptosis is first initiated, followed by a temporal induction of necrosis and lysis of the host upon growth transition into the postexponential phase (Alli et al, 2000;Byrne & Swanson, 1998;Kirby et al, 1998). The pore-forming activity mediates lysis of the host cell, and mutants defective in pore-forming activity are defective in lysis of the host cell and are delayed in subsequent egress from mammalian (Alli et al, 2000) and protozoan cells (Gao & Abu Kwaik, 2000b).…”