Genetic susceptibility and caspase activation in mouse and human macrophages are distinct for Legionella longbeachae and L. pneumophila

Abstract

Legionella pneumophila is the predominant cause of Legionnaires' disease in the United States and Europe, while Legionella longbeachae is the common cause of the disease in Western Australia. Although clinical manifestations by both intracellular pathogens are very similar, recent studies have shown that phagosome biogeneses of both species within human macrophages are distinct (R. Asare and Y. Abu Kwaik, Cell. Microbiol., in press). Most inbred mouse strains are resistant to infection by L. pneumophila, with the exception of the A/J mouse strain, and this genetic susceptibility is associated with polymorphism in the naip5 allele and flagellin-mediated early activation of caspase 1 and pyropoptosis in nonpermissive mouse macrophages. Here, we show that genetic susceptibility of mice to infection by L. longbeachae is independent of allelic polymorphism of naip5. L. longbeachae replicates within bone marrow-derived macrophages and in the lungs of A/J, C57BL/6, and BALB/c mice, while L. pneumophila replicates in macrophages in vitro and in the lungs of the A/J mouse strain only. Quantitative real-time PCR studies on infected A/J and C57BL/6 mouse bone marrow-derived macrophages show that both L. longbeachae and L. pneumophila trigger similar levels of naip5 expression, but the levels are higher in infected C57BL/6 mouse macrophages. In contrast to L. pneumophila, L. longbeachae has no detectable pore-forming activity and does not activate caspase 1 in A/J and C57BL/6 mouse or human macrophages, despite flagellation. Unlike L. pneumophila, L. longbeachae triggers only a modest activation of caspase 3 and low levels of apoptosis in human and murine macrophages in vitro and in the lungs of infected mice at late stages of infection. We conclude that despite flagellation, infection by L. longbeachae is independent of polymorphism in the naip5 allele and L. longbeachae does not trigger the activation of caspase 1, caspase 3, or late-stage apoptosis in mouse and human macrophages. Neither species triggers caspase 1 activation in human macrophages.

FIG. 1.

L. longbeachae strains replicate equally in immortalized bone marrow-derived macrophages from A/J, C57BL/6, and BALB/c mice. Immortalized murine macrophages were infected with L. longbeachae strain D4968 at an MOI of 10 for 1 h, followed by gentamicin treatment for 1 h. The number of CFU of L. longbeachae was enumerated over 48 h. The results are representative of three independent experiments, and error bars represent standard deviations.

FIG. 2.

Infection of A/J, C57BL/6, and BALB/c mice with L. longbeachae and L. pneumophila. (A) A/J mice were infected intratracheally with different doses of the L. longbeachae clinical isolate D4968 or (B) L. pneumophila. (C) A/J, C57BL/6, and BALB/c mice were infected with 1 × 10⁵ CFU of L. longbeachae strain D4968, and lethality was monitored over 14 days.

FIG. 3.

Inbred mouse strains are equally susceptible to infection by different strains of L. longbeachae. Intrapulmonary replication of L. longbeachae and L. pneumophila in A/J, C57BL/6, and BALB/c mice was determined. The three strains of mice (n = 12 each) were inoculated with (A) 1 × 10⁵ CFU of the L. longbeachae clinical isolate D4968, (B) 1 × 10⁵ CFU of the L. longbeachae clinical isolate D4973, and (C) 10⁶ CFU of L. pneumophila. Lungs were harvested, and the number of CFU in the lungs was determined at the indicated time points. The results are representative of three independent experiments, and error bars represent standard deviations.

FIG. 4.

High level of expression of naip5 in C57BL/6 macrophages compared to that in A/J mouse macrophages. Immortalized bone marrow-derived macrophages from A/J and C57BL/6 mice were infected with L. longbeachae or L. pneumophila at an MOI of 10, and total RNA was isolated from macrophages at 2, 6, and 12 h postinfection. The levels of naip5 expression in infected as well as uninfected cells were determined by quantitative reverse transcriptase PCR. Levels of expression of naip5 were determined relative to those in uninfected cells (A and B) and are also expressed as ratios of C57BL/6 to A/J mouse macrophages (C). The results are representative of three independent experiments, and the error bars represent standard deviations.

FIG. 5.

Flagellar expression by L. longbeachae during the intracellular infection. Representative images of U937 human macrophages infected with the L. longbeachae clinical isolate D4968 and ATCC strain 33462 at 36 h postinfection. The expression of flagella was determined using anti (α)-flagellin antibody against flagellin and compared to that in L. pneumophila-infected cells. The results are representative of two independent experiments.

FIG. 6.

L. longbeachae does not induce caspase 1 activation in mBDMs or hMDMs. A/J and BALB/c mouse macrophages and hMDMs were infected with the L. longbeachae clinical isolate D4968 and ATCC 33462, and the activation of caspase 1 (C1) and apoptosis (TUNEL [T]) were determined at 2, 8, and 18 h postinfection. L. pneumophila and its isogenic dotA mutant were used as positive and negative controls, respectively, and simvastatin was used as a positive control for caspase 1 in human macrophages. Francisella tularensis (Ft) was used as a positive control for hMDMs. The data are representative of three independent experiments.

FIG. 7.

Detection of caspase 3 activation by its cleavage of a fluorescent substrate. (A) Human U937 macrophages were infected with the L. longbeachae clinical isolate D4968 and L. pneumophila, and the number of CFU was monitored over 48 h. (B) Human U937 macrophages were infected with the L. longbeachae clinical isolate D4968, D4969, or D4973, L. pneumophila, or its dotA isogenic mutant. The caspase 3 activity to cleave the fluorescent substrate was measured at 6 h postinfection and expressed as arbitrary fluorescence units (AFU). The data are representative of three independent experiments, and the error bars represent standard deviations.

FIG. 8.

Single-cell analysis of caspase 3 activation in human macrophages. hMDMs were infected with the L. longbeachae clinical isolate D4968, L. pneumophila, or its dotA isogenic mutant at an MOI of 10, and caspase 3 activation was analyzed at 6 h and 24 h postinfection. Bacteria were detected by a specific antibody, while active caspase 3 was detected by an antiactive caspase 3 antibody. (A) Representative confocal microscopy images at several time points are shown. (B) Quantification of the percentage of cells with active caspase 3 was determined by analysis of 100 infected cells from three different coverslips in each experiment. There were equivalent numbers of bacteria per phagosome for both bacterial species. The data are representative of three independent experiments, and the error bars represent standard deviations.

FIG. 9.

L. longbeachae triggers low caspase 3 activation in murine macrophages. A/J and BALB/c mouse macrophages were infected with the L. longbeachae clinical isolate D4968 and ATCC 33462, and levels of caspase 3 activation (C3) and apoptosis (TUNEL [T]) were determined at 2, 8, and 18 h postinfection. L. pneumophila and its isogenic dotA mutant were used as positive and negative controls, respectively. The data are representative of three experiments.

FIG. 10.

L. longbeachae induces low levels of late-stage apoptosis in human macrophages. hMDMs were infected with L. longbeachae strain D4968, L. pneumophila, or its dotA isogenic mutant at an MOI of 10, and the level of apoptosis in infected cells was analyzed at 6 and 24 h postinfection. The bacteria were labeled with an antibody, while apoptotic nuclei were detected by TUNEL. Representative images at the indicated time points are shown in panel A. (B) Quantification of the percentage of apoptotic cells was determined by analysis of 100 infected cells from three different coverslips in each experiment. The data are representative of three independent experiments, and the error bars represent standard deviations.

FIG. 11.

L. longbeachae induces low levels of pulmonary apoptosis in A/J mice. A/J mice were infected with 10⁶ CFU of L. longbeachae strain D4968 or L. pneumophila. Lungs from the infected mice were harvested at 24, 48, and 72 h and stained for TUNEL to detect apoptotic nuclei. Only the 48-h time point is shown for L. pneumophila because the results from all the time points are similar. The images are representative of 20 different microscopic fields from the lungs of three animals for each time point. The results are representative of three independent experiments.