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/Icm-dependent 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.

FIG. 1.

Effect of preinduction of caspase-3 activity on intracellular replication of icmS, icmR, and icmQ mutants in U937 macrophages. The graphs show the growth kinetics of icmR (A), icmQ (B), icmS (C), A100 (D), and dotA strains in untreated U937 macrophages or macrophages treated with 0.5 or 1.0 μM staurosporin (St.) for 1 h prior to infection. Infections were performed for 1 h using an MOI of 10 (see Materials and Methods). The cells were lysed at different time intervals, and the numbers of bacteria in the monolayers were enumerated after growth on agar plates. The results are representative of three independent experiments. The experiments were done in triplicate, and error bars represent standard deviations (some of the error bars are too small to be displayed).

FIG. 2.

Kinetics of staurosporin-induced caspase-3 activity and growth kinetics of AA100 in staurosporin-treated U937 macrophages. (A) Kinetics of caspase-3 activity in U937 cells treated with 0.5 and 1.0 μM staurosporin (St.) for different time periods. Caspase-3 activity was determined using a fluorogenic substrate specific for caspase-3 and is expressed in AFUs. (B) Growth kinetics of AA100 in U937 macrophages treated with either 0.5 or 1.0 μM staurosporin for 1 h prior to infection. Infections were performed for 1 h using an MOI of 10 (see Materials and Methods). The cells were lysed at different time intervals, and the numbers of bacteria in the monolayers were enumerated after growth on agar plates. The results are representative of three independent experiments. The experiments were done in triplicate, and error bars represent standard deviations.

FIG. 3.

TNF-α-mediated preactivation of caspase-3 blocks intracellular replication of AA100 in U937 macrophages. (A) TNF-α-induced caspase-3 activity in U937 macrophages in the presence of different concentrations of cycloheximide (CHX). Caspase-3 activity was determined using a fluorogenic substrate specific for caspase-3 and is expressed in AFUs. (B) Growth kinetics of AA100 and the dotA mutant in untreated macrophages and of AA100 in macrophages treated with TNF-α-cycloheximide for 5 h prior to infection. Infections were performed for 1 h using an MOI of 10 (as described in Materials and Methods). The cells were lysed at different time intervals, and the numbers of bacteria in the monolayers were enumerated after growth on agar plates. The results are representative of three independent experiments. The experiments were done in triplicate, and error bars represent standard deviations. Some of the error bars are too small to be displayed.

FIG. 4.

Effect of preactivation of caspase-3 on intracellular replication of AA100 in mouse macrophages. (A) Caspase-3 activity in J774A.1 macrophages at 6 h postinfection with AA100 or the dotA mutant using an MOI of 10 for 1 h compared to that after treatment with staurosporin (St.). (B) Kinetics of caspase-3 activity in J774A.1 macrophages at different time points after treatment with staurosporin. Caspase-3 activity was determined using a fluorogenic substrate specific for caspase-3 and is expressed in AFUs. (C) Growth kinetics of AA100 and the dotA mutant in untreated J774A.1 macrophages and of AA100 in J774A.1 macrophages treated with staurosporin for 2 h prior to infection at an MOI of 10 for 1 h. The cells were lysed at different time intervals, and the numbers of bacteria in the monolayers were enumerated after growth on agar plates. The results are representative of three independent experiments. The experiments were done in triplicate, and error bars represent standard deviations. Some of the error bars are too small to be displayed.

FIG. 5.

Effect of caspase-3 activation during early stages on exponential replication of L. pneumophila. The graphs show the amount of intracellular replication of AA100 in U937 macrophages that were either untreated or treated at 8 h postinfection with 0.5 μM staurosporin (St.) (A) or TNF-α-cycloheximide (CHX) (B). Infections were performed for 1 h using an MOI of 10 (see Materials and Methods). The cells were lysed at different time intervals, and the numbers of bacteria in the monolayers were enumerated after growth on agar plates. The results are representative of three independent experiments. The experiments were done in triplicate, and error bars represent standard deviations. Some of the error bars are too small to be displayed.

FIG. 6.

Kinetics of caspase-3 activation and apoptosis induction in L. pneumophila-infected and staurosporin-treated U937 macrophages. L. pneumophila-infected cells (MOI of 5 for 1 h) were fixed and labeled 2 to 18 h after infection. Staurosporin-treated cells were fixed and labeled 4, 8, and 12 h after treatment. (A) For each time point, ∼100 macrophages were analyzed by laser scanning confocal microscopy for the number of intracellular bacteria, for active caspase-3 (C3), and for apoptotic nuclei detected by TUNEL (TU) assays. (B) AFUs of caspase-3 activity in nonapoptotic L. pneumophila-infected cells compared to those in cells treated with 1 μM staurosporin. (C) Percentages of apoptotic nuclei in cells treated with different concentrations of staurosporin. (D) Average AFUs/cell for active caspase-3. The experiments were done in triplicate, and the results are representative of three independent experiments.

FIG. 7.

Representative laser scanning confocal microscopy images of L. pneumophila-infected macrophages. U937 macrophages were infected with the parental strain AA100 at an MOI of 5. Infected macrophages were fixed and permeabilized 2, 4, 8, 12, and 18 h after infection. Apoptotic nuclei were labeled using TUNEL (green), intracellular bacteria were labeled using mouse monoclonal anti-L. pneumophila antibodies followed by Alexa fluor 555-conjugated secondary antibodies (red), and active caspase-3 was labeled using rabbit polyclonal anti-active caspase-3 antibodies followed by Alexa fluor 647-conjugated secondary antibodies (blue). The experiments were done in triplicate, and the results are representative of three independent experiments.