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. 2010 Jan;78(1):423-32.
doi: 10.1128/IAI.00070-09. Epub 2009 Oct 19.

Mouse macrophages are permissive to motile Legionella species that fail to trigger pyroptosis

Natalie N Whitfield  1 , Brenda G Byrne, Michele S Swanson
Affiliations

Mouse macrophages are permissive to motile Legionella species that fail to trigger pyroptosis

Natalie N Whitfield et al. Infect Immun. 2010 Jan.

Abstract

Legionella pneumophila, a motile opportunistic pathogen of humans, is restricted from replicating in the lungs of C57BL/6 mice. Resistance of mouse macrophages to L. pneumophila depends on recognition of cytosolic flagellin. Once detected by the NOD-like receptors Naip5 and Ipaf (Nlrc4), flagellin triggers pyroptosis, a proinflammatory cell death. In contrast, motile strains of L. parisiensis and L. tucsonensis replicate profusely within C57BL/6 macrophages, similar to flagellin-deficient L. pneumophila. To gain insight into how motile species escape innate defense mechanisms of mice, we compared their impacts on macrophages. L. parisiensis and L. tucsonensis do not induce proinflammatory cell death, as measured by lactate dehydrogenase (LDH) release and interleukin-1beta (IL-1beta) secretion. However, flagellin isolated from L. parisiensis and L. tucsonensis triggers cell death and IL-1beta secretion when transfected into the cytosol of macrophages. Neither strain displays three characteristics of the canonical L. pneumophila Dot/Icm type IV secretion system: sodium sensitivity, LAMP-1 evasion, and pore formation. Therefore, we postulate that when L. parisiensis and L. tucsonensis invade a mouse macrophage, flagellin is confined to the phagosome, protecting the bacteria from recognition by the cytosolic surveillance system and allowing Legionella to replicate. Despite their superior capacity to multiply in mouse macrophages, L. parisiensis and L. tucsonensis have been associated with only two cases of disease, both in renal transplant patients. These results point to the complexity of disease, a product of the pathogenic potential of the microbe, as defined in the laboratory, and the capacity of the host to mount a measured defense.

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Figures

FIG. 1.
FIG. 1.
Flagellated strains of L. parisiensis and L. tucsonensis evade C57BL/6 macrophage restriction of replication. The intracellular growth in C57BL/6 macrophages of L. pneumophila (○), flaA mutant L. pneumophila (□), L. parisiensis (⋄), and L. tucsonensis (▵) was determined by quantifying CFU at the times shown and dividing by the CFU associated with cells at 2 h. The values shown are the means ± standard deviations (SD) calculated for triplicate samples in one experiment that is representative of three or more independent trials.
FIG. 2.
FIG. 2.
L. parisiensis and L. tucsonensis strains do not trigger cell death or IL-1B secretion. (A) C57BL/6 macrophages were incubated for 1 h with twofold dilutions of L. pneumophila (⧫), flaA mutant L. pneumophila (▪), L. parisiensis (▴), and L. tucsonensis (•), and viability was quantified by Alamar Blue reduction. Results from one experiment, representative of three, are shown. (B) Macrophages pretreated with LPS were infected with the strain indicated for 2 h, and secreted IL-1β was quantified. Results shown are means ± standard errors (SE) for duplicate wells in three experiments.
FIG. 3.
FIG. 3.
Characterization of crude flagellin preparations. Flagellin samples prepared in parallel from broth cultures were separated by SDS-PAGE and analyzed by Coomassie blue staining (A) of L. pneumophila (lane 2), flaA mutant L. pneumophila (lane 3), L. parisiensis (lane 4), and L. tucsonensis (lane 5) or Western analysis, using anti-L. pneumophila flagellin antibody (B), of L. pneumophila (lane 1), flaA mutant L. pneumophila (lane 2), L. parisiensis (lane 3), and L. tucsonensis (lane 4). Positions of molecular mass standards are shown (lane 1 in panel A). (C) Relative genomic hybridization under low-stringency conditions with DNA probes for the indicated regions of L. pneumophila flaA.
FIG. 4.
FIG. 4.
Flagellin preparations from L. parisiensis and L. tucsonensis trigger macrophage cell death. To determine whether flagellins from L. parisiensis and L. tucsonensis trigger pyroptosis, the amounts of LDH (A) and IL-1β (B) released from C57BL/6 macrophages transfected with heat-treated crude flagellin (1.25 μg; black bars) or treated with proteinase K (gray bars) and incubated for 2 h were quantified. The data shown are means ± SE for three experiments, where asterisks indicate statistical differences from flaA mutant L. pneumophila (P < 0.05 using one-way ANOVA). (C) To determine whether the pyroptosis response to cytosolic flagellin required the NLR proteins Naip5 and Ipaf, LDH released from macrophages derived from C57BL/6 or Naip5−/− Ipaf−/− double mutant mice was quantified as in panel A. Asterisks indicate statistical differences (P < 0.01) compared to C57BL/6 macrophages exposed to WT L. pneumophila, calculated using one-way ANOVA; the carat indicates that P = 0.016.
FIG. 5.
FIG. 5.
L. parisiensis and L. tucsonensis do not display features of the canonical Dot/Icm type IV secretion system. (A) Sodium sensitivity was quantified by plating E- or PE-phase broth cultures on medium with or without 100 mM NaCl and using the formula [(CFU on CYET+NaCl)/(CFU on CYET)] × 100. (B) Colocalization with the late endosomal and lysosomal protein LAMP-1 was quantified by immunofluorescence microscopy after incubating C57BL/6 macrophages for 1.5 h with PE-phase WT L. pneumophila, dotA mutant L. pneumophila, L. parisiensis, or L. tucsonensis. Results shown are means ± SE for three experiments. (C) Permeability of A/J macrophages after incubation for 1 h with PE-phase Legionella was determined by uptake of ethidium bromide. The data shown are means ± SE calculated from three experiments.
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