The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila

Abstract

The ability of Legionella pneumophila to cause legionnaires' disease is dependent on its capacity to replicate within cells in the alveolar spaces. The bacteria kill mammalian cells in two phases: induction of apoptosis during the early stages of infection, followed by an independent and rapid necrosis during later stages of the infection, mediated by a pore-forming activity. In the environment, L. pneumophila is a parasite of protozoa. The molecular mechanisms by which L. pneumophila kills the protozoan cells, after their exploitation for intracellular proliferation, are not known. In an effort to decipher these mechanisms, we have examined induction of both apoptosis and necrosis in the protozoan Acanthamoeba polyphaga upon infection by L. pneumophila. Our data show that, although A. polyphaga undergoes apoptosis following treatment with actinomycin D, L. pneumophila does not induce apoptosis in these cells. Instead, intracellular L. pneumophila induces necrotic death in A. polyphaga, which is mediated by the pore-forming activity. Mutants of L. pneumophila defective in expression of the pore-forming activity are indistinguishable from the parental strain in intracellular replication within A. polyphaga. The parental strain bacteria cause necrosis-mediated lysis of all the A. polyphaga cells within 48 h after infection, and all the intracellular bacteria are released into the tissue culture medium. In contrast, all cells infected by the mutants remain intact, and the intracellular bacteria are 'trapped' within A. polyphaga after the termination of intracellular replication. Failure to exit the host cell after termination of intracellular replication results in a gradual decline in the viability of the mutant strain bacteria within A. polyphaga starting 48h after infection. Our data show that the pore-forming activity of L. pneumophila is not required for intracellular bacterial replication within A. polyphaga but is required for killing and exiting the protozoan host upon termination of intracellular replication.

Enhanced survival but not amplification of Francisella spp. in the presence of free-living amoebae

“…This is in contrast to the water-based pathogen, Lp, which exhibits high infectivity potential for Acanthamoeba spp. and V. vermiformis resulting in rapid intracellular amplification and release of Lp and significant amoebal lysis ( Figure 5) [32,[42][43][44][45]. The isolation and identification of amoebae-resisting bacteria (ARB) using the well-established co-culture method involves inoculation of samples onto a monolayer of axenic amoebae and continuous monitoring of cultures for amoebal lysis, resulting from infection by ARBs (reviewed in [46,47]).…”

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).…”

Legionella pneumophila glucosyltransferase inhibits host elongation factor 1A

“…These organellae are associated with ribosome-studded membranes and support multiplication of the bacteria (6,7). Eventually, host cells die because of induction of apoptosis or necrosis (8)(9)(10), and the pathogens are released to invade new pools of phagocytes of the host (10,11). During recent years several genetic loci and bacterial factors have been identified that are suggested to be implicated in Legionella-host cell interactions (12).…”