Induction of Rapid Cell Death by an Environmental Isolate of Legionella pneumophila in Mouse Macrophages

Intracellular growth of strain LPE509 in three different hosts. Differentiated human D. discoideum (A), A. castellanii (B), U937 macrophage (C), or bone marrow-derived macrophages (D) from A/J mouse were infected with indicated bacterial strains grown to post-exponential phase at an MOI of 0.05. Infections were synchronized 2 h after adding the bacteria to the cells. At the indicated time points, infected samples were lysed with saponin, and appropriately diluted cell lysates were plated onto bacteriological charcoal yeast extract medium. All infections were performed in triplicate, and the data shown were from one representative experiment of four experiments with highly similar results.

Intracellular trafficking of strain LPE509 in A/J macrophages. Bone marrow-derived macrophages seeded on glass coverslips were infected with properly grown bacterial strains at an MOI of 2. One hour after infection, samples were washed with medium and were fixed at the indicated time points. After labeling extracellular and intracellular bacteria differently by immunostaining, the host proteins LAMP-1 (A) and Rab1 (B) were stained with specific antibodies. The data were collected using an IX-81 Olympus microscope by inspecting bacterial vacuoles for the association of the protein staining signals. All infections were performed in triplicate, and at least 100 bacterial phagosomes were scored for each sample. The data shown were the averages of two independent experiments.

Formation of replicative vacuoles by strain LPE509 in macrophages. A/J mouse macrophages on glass coverslips were infected with appropriately grown bacterial strains at an MOI of 1. Infections were synchronized 1 h after bacterial challenge and were allowed to proceed for an additional 13 h. Samples were then fixed and subjected to immunostaining to label intracellular and extracellular L. pneumophila with different fluorescent colors. When necessary, samples were further staining with DAPI (4′,6′-diamidino-2-phenylindole) to highlight the nuclei of host cells. The bacterial phagosomes were scored under a fluorescence microscope according to the number of bacteria they harbored. Phagosomes containing more than 10 bacteria were categorized as large; those with 4 to 9 bacteria were called medium and those with 1 to 3 bacteria were classified as small vacuoles. (A) The distributions of vacuole sizes of strain JR32 and LPE509 and their derivatives defective in the Dot/Icm transporter. **, P < 0.001 for values of big or small vacuoles between strain JR32 and LPE509. (B) Representative images of large vacuoles by both bacterial strains. Note the condensed nucleus of the cell infected by strain LPE509. Bar, 10 μm.

Intracellular growth of a mutant of strain LPE509 lacking flagellin in A/J macrophages. (A) Expression of flagellin by the testing bacterial strains. Cells of indicated strains grown to post-exponential phase were lysed with Laemmli sample buffer and total protein was resolved by SDS-PAGE. Flagellin was detected by a specific antibody and the isocitrate dehydrogenase (ICDH) was probed as a loading control (lower panel). (B) Testing bacterial strains grown in AYE broth to post-exponential phase were used to infect bone marrow-derived macrophages from A/J mice at an MOI of 0.05. Infections synchronized 2 h after adding bacteria were lysed with saponin at indicated time points, and total bacterial counts were determined by plating appropriate diluted lysates onto bacteriological medium. Infections were performed in triplicate, and similar results were obtained in three independent experiments.

Infection by strain LPE509 damages macrophage morphology and membrane integrity. Bone marrow derived macrophages from A/J mice were challenged with indicated bacterial strains grown to post-exponential phase at an MOI of 1 for 2 h. (A and B) Cell morphology and membrane integrity of infected samples was examined under a microscope. Similarly infected samples were simultaneously stained with PI and Hoechst 33342 and were analyzed by imaging with a fluorescence microscope. Bar, 10 μm. (C) Quantitation of membrane damaged cells. Membrane damage was determined by positive staining by PI under a microscope, at least 500 cells were scored in each sample for infections done in triplicate. The data shown are from one representative experiment from three independent experiments with similar results.

Infection by LPE509 caused extensive cell death on A/J macrophages. (A and B) Bone marrow-derived macrophages from A/J mice were infected by indicated L. pneumophila strains at an MOI of 1 for 14 h, and infected cells were immunostained to identify extracellular and intracellular bacteria, followed by TUNEL staining to obtain representative images of the nuclei of infected cells. The label of host nuclei by the TUNEL reagent was inspected and scored under a fluorescence microscope. Bar, 10 μm. (C) Cells infected with the similar bacterial strains at an MOI of 1 were assayed for the release of LDH. Each experiment was performed in triplicate, and similar results were obtained in four independent experiments.