Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles

Abstract

After ingestion by macrophages, Legionella pneumophila inhibits acidification and maturation of its phagosome. After a 6-10-h lag period, the bacteria replicate for 10-14 h until macrophage lysis releases dozens of progeny. To examine whether the growth phase of intracellular L. pneumophila determines the fate of its phagosome, interactions between the endosomal network and pathogen vacuoles were analyzed throughout the primary infection period. Surprisingly, as L. pneumophila replicated exponentially, a significant proportion of the vacuoles acquired lysosomal characteristics. By 18 h, 70% contained lysosomal-associated membrane protein 1 (LAMP-1) and 40% contained cathepsin D; 50% of the vacuoles could be labeled by endocytosis, and the pH of this population of vacuoles averaged 5.6. Moreover, L. pneumophila appeared to survive and replicate within lysosomal compartments: vacuoles harboring more than five bacteria also contained LAMP-1, inhibition of vacuole acidification and maturation by bafilomycin A1 inhibited bacterial replication, bacteria within endosomal vacuoles responded to a metabolic inducer by expressing a gfp reporter gene, and replicating bacteria obtained from macrophages, but not broth, were acid resistant. Understanding how L. pneumophila first evades and then exploits the endosomal pathway to replicate within macrophages may reveal the mechanisms governing phagosome maturation, a process also manipulated by Mycobacteria, Leishmania, and Coxiella.

Figure 1

The acquisition of endosomal proteins by L. pneumophila vacuoles. Macrophages infected with L. pneumophila for the periods indicated were labeled with antibody specific for LAMP-1 or cathepsin D, then stained with DAPI. (A) At 8 h, L. pneumophila vacuoles rarely colocalized with LAMP-1 or cathepsin D (black arrowheads); after 16 h, many L. pneumophila vacuoles were ringed by LAMP-1 and contained cathepsin D (white arrowheads). The percentage of L. pneumophila vacuoles that contained either LAMP-1 (B) or cathepsin D (C) at the time indicated was scored in three separate experiments, each represented by a different symbol (circles, squares, or diamonds).

Figure 1

The acquisition of endosomal proteins by L. pneumophila vacuoles. Macrophages infected with L. pneumophila for the periods indicated were labeled with antibody specific for LAMP-1 or cathepsin D, then stained with DAPI. (A) At 8 h, L. pneumophila vacuoles rarely colocalized with LAMP-1 or cathepsin D (black arrowheads); after 16 h, many L. pneumophila vacuoles were ringed by LAMP-1 and contained cathepsin D (white arrowheads). The percentage of L. pneumophila vacuoles that contained either LAMP-1 (B) or cathepsin D (C) at the time indicated was scored in three separate experiments, each represented by a different symbol (circles, squares, or diamonds).

Figure 1

The acquisition of endosomal proteins by L. pneumophila vacuoles. Macrophages infected with L. pneumophila for the periods indicated were labeled with antibody specific for LAMP-1 or cathepsin D, then stained with DAPI. (A) At 8 h, L. pneumophila vacuoles rarely colocalized with LAMP-1 or cathepsin D (black arrowheads); after 16 h, many L. pneumophila vacuoles were ringed by LAMP-1 and contained cathepsin D (white arrowheads). The percentage of L. pneumophila vacuoles that contained either LAMP-1 (B) or cathepsin D (C) at the time indicated was scored in three separate experiments, each represented by a different symbol (circles, squares, or diamonds).

Figure 3

The accessibility of L. pneumophila vacuoles to endocytic probes. Macrophages incubated with either formaldehyde-fixed E. coli for 1 h or L. pneumophila (Lp) for the period indicated were incubated with TR-OV for 15 min, then in fresh medium for an additional 5, 30, or 120 min. After fixation and DAPI staining, blinded samples were scored for the percentage of vacuoles that had accumulated detectable TR-OV. Each bar represents the cumulative results from three separate experiments in which 50 vacuoles per condition were scored.

Figure 2

The acquisition of fluid-phase lysosomal markers by L. pneumophila vacuoles. Macrophages whose lysosomes were prelabeled by endocytosis of TR-OV or TR-DEX were infected with L. pneumophila for the period indicated, fixed, incubated with DAPI to label the bacteria, then analyzed microscopically. (A) At 8 h, bacterial vacuoles were isolated from the endocytic network (black arrowheads); by 16 h, the lumen of many vacuoles harboring L. pneumophila contained a fluorescent probe (white arrowheads). The 24-h panel reveals two separate vacuoles heavily laden with bacteria; the one at left had obviously fused (white arrowheads), whereas TR-OV accumulation by the vacuole at right was below the limit of detection (black arrowheads). (B) The percentage of 50 vacuoles that contained either TR-OV (filled symbols) or TR-DEX (open symbols) at the time indicated was scored by fluorescence microscopy in four separate experiments, each represented by a different symbol (diamonds or squares).

Figure 2

The acquisition of fluid-phase lysosomal markers by L. pneumophila vacuoles. Macrophages whose lysosomes were prelabeled by endocytosis of TR-OV or TR-DEX were infected with L. pneumophila for the period indicated, fixed, incubated with DAPI to label the bacteria, then analyzed microscopically. (A) At 8 h, bacterial vacuoles were isolated from the endocytic network (black arrowheads); by 16 h, the lumen of many vacuoles harboring L. pneumophila contained a fluorescent probe (white arrowheads). The 24-h panel reveals two separate vacuoles heavily laden with bacteria; the one at left had obviously fused (white arrowheads), whereas TR-OV accumulation by the vacuole at right was below the limit of detection (black arrowheads). (B) The percentage of 50 vacuoles that contained either TR-OV (filled symbols) or TR-DEX (open symbols) at the time indicated was scored by fluorescence microscopy in four separate experiments, each represented by a different symbol (diamonds or squares).

Figure 4

The bacterial density in vacuoles that contained LAMP-1 (black bars) or lacked LAMP-1 (white bars). (A) Macrophages infected for the period indicated were fixed and processed for microscopic localization of LAMP-1 and L. pneumophila. At the times indicated, vacuoles were scored for the presence of LAMP-1 and the number of bacteria. As it was not possible to count accurately >20 bacteria in one compartment, these were scored as >20. (B) After a 1-h infection, macrophages were treated with the proton ATPase inhibitor BFA, incubated for the times indicated, then processed as described above. In each of three independent experiments, 50 vacuoles were analyzed for each condition at the times indicated. The population of vacuoles analyzed for A was the same as that depicted in Fig. 1 B; the population of vacuoles analyzed for B was the same as that represented by Fig. 7.

Figure 6

The inhibition of L. pneumophila vacuole maturation by BFA. Macrophage lysosomes were labeled either with antibody specific for LAMP-1 or by endocytosis of TR-OV. 1 h after infection, 25 nM BFA was added to the culture medium for the duration of the experiment (open symbols) or was not (filled symbols). (A) In the presence of BFA, L. pneumophila vacuoles failed to merge with the endocytic network (black arrowheads), and each typically contained <15 bacteria. The percentage of 50 vacuoles that colocalized with either (B) LAMP-1 or (C) TR-OV was scored by fluorescence microscopy over the course of a primary infection in three separate experiments, each represented by a different symbol (circles, squares, or diamonds).

Figure 6

The inhibition of L. pneumophila vacuole maturation by BFA. Macrophage lysosomes were labeled either with antibody specific for LAMP-1 or by endocytosis of TR-OV. 1 h after infection, 25 nM BFA was added to the culture medium for the duration of the experiment (open symbols) or was not (filled symbols). (A) In the presence of BFA, L. pneumophila vacuoles failed to merge with the endocytic network (black arrowheads), and each typically contained <15 bacteria. The percentage of 50 vacuoles that colocalized with either (B) LAMP-1 or (C) TR-OV was scored by fluorescence microscopy over the course of a primary infection in three separate experiments, each represented by a different symbol (circles, squares, or diamonds).

Figure 6

The inhibition of L. pneumophila vacuole maturation by BFA. Macrophage lysosomes were labeled either with antibody specific for LAMP-1 or by endocytosis of TR-OV. 1 h after infection, 25 nM BFA was added to the culture medium for the duration of the experiment (open symbols) or was not (filled symbols). (A) In the presence of BFA, L. pneumophila vacuoles failed to merge with the endocytic network (black arrowheads), and each typically contained <15 bacteria. The percentage of 50 vacuoles that colocalized with either (B) LAMP-1 or (C) TR-OV was scored by fluorescence microscopy over the course of a primary infection in three separate experiments, each represented by a different symbol (circles, squares, or diamonds).

Figure 5

The inhibition of L. pneumophila growth in macrophages by the proton ATPase inhibitor BFA. Infected macrophages were incubated with 25 nM BFA (open circles) or without drug (filled squares), and the yield of CFU was determined at the times indicated. Shown are the means and SE values calculated from four separate experiments, each performed in triplicate.

Figure 7

GFP expression by bacteria within endocytic vacuoles. Macrophages infected for 16 h with L. pneumophila carrying pMMB-GRN were incubated with IPTG to induce GFP expression and with TR-OV to label the lysosomes. Intracellular bacteria were identified by DAPI staining and scored for colocalization with the lysosomal probe and for expression of GFP. (A) The majority of lysosomal pathogen vacuoles harbored GFP-expressing L. pneumophila, (B) whereas a minority of the endosomal vacuoles contained bacteria that exhibited only DAPI fluorescence.

Figure 8

Acid tolerance of L. pneumophila isolated from macrophages. Macrophages infected with L. pneumophila for 3, 12, or 18 h were lysed, and the bacteria were transferred to broth of pH 5.0 or 6.9. In parallel, E or PE L. pneumophila from pH 6.9 broth were diluted into broth of pH 5.0 or 6.9. After 4 h, samples were plated on CYET to quantify CFU. Percentage survival was calculated as (CFU/ml of AYET, pH 5.0)/(CFU/ml of AYET, pH 6.9) × 100. Means and SE were calculated from four separate experiments.