Incomplete Activation of Macrophage Apoptosis during Intracellular Replication of Legionella pneumophila

Abstract

The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icmdependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.

Legionella pneumophila inhibits macrophage apoptosis by targeting pro-death members of the Bcl2 protein family

“…2). Consistent with earlier studies (16), cells infected by the wild-type strain were resistant to this cell death stimulus, with only Ϸ26% of the cells being apoptotic (Fig. 2).…”

“…Macrophages harboring replicating L. pneumophila exhibit resistance to apoptosis stimuli (16). To examine whether SidF contributes to such resistance, we infected U937 cells with L. pneumophila and subsequently treated these cells with staurosporine for 4 h. The apoptotic status of infected cells was examined by Hoechst staining followed by microscopic inspection.…”

“…However, recent studies indicate that vigorous bacterial replication did not lead to apoptosis in infected cells (15). Instead, macrophages harboring actively replicating L. pneumophila exhibit strong resistance to exogenous cell death stimuli (16). These observations indicate that an apoptotic process is initiated in macrophages in response to L. pneumophila infection and replication, but the bacteria are able to inhibit or reverse the process in a Dot/Icm-dependent manner, possibly by injecting effectors that can interfere with apoptotic pathways.…”

A Legionella pneumophila -translocated substrate that is required for growth within macrophages and protection from host cell death

“…As there is evidence for Dot/Icm-dependent caspase activation (20), macrophages incubated with bacteria were probed with FITC-VAD-FMK, a fluorescent pan-caspase activation marker. Association of cells with L. pneumophila sdhA Ϫ resulted in an increase in the number of macrophages reacting with the probe relative to that observed in macrophages incubated with WT bacteria (Fig.…”

“…The translocated substrates of the Dot/Icm system stimulate host cell death by multiple pathways (18)(19)(20). At a high multiplicity of infection (moi), target macrophages undergo rapid cell death, whereas at low moi, a fraction of host cells undergo apoptotic death (19,21,22).…”