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. 2016 Jan 7;11(1):e0146410.
doi: 10.1371/journal.pone.0146410. eCollection 2016.

The Sphingosine-1-Phosphate Lyase (LegS2) Contributes to the Restriction of Legionella pneumophila in Murine Macrophages

Arwa Abu Khweek  1 , Apurva Kanneganti  2 , Denis C Guttridge D  3 , Amal O Amer  2
Affiliations

The Sphingosine-1-Phosphate Lyase (LegS2) Contributes to the Restriction of Legionella pneumophila in Murine Macrophages

Arwa Abu Khweek et al. PLoS One. .

Abstract

L. pneumophila is the causative agent of Legionnaires' disease, a human illness characterized by severe pneumonia. In contrast to those derived from humans, macrophages derived from most mouse strains restrict L. pneumophila replication. The restriction of L. pneumophila replication has been shown to require bacterial flagellin, a component of the type IV secretion system as well as the cytosolic NOD-like receptor (NLR) Nlrc4/ Ipaf. These events lead to caspase-1 activation which, in turn, activates caspase-7. Following caspase-7 activation, the phagosome-containing L. pneumophila fuses with the lysosome, resulting in the restriction of L. pneumophila growth. The LegS2 effector is injected by the type IV secretion system and functions as a sphingosine 1-phosphate lyase. It is homologous to the eukaryotic sphingosine lyase (SPL), an enzyme required in the terminal steps of sphingolipid metabolism. Herein, we show that mice Bone Marrow-Derived Macrophages (BMDMs) and human Monocyte-Derived Macrophages (hMDMs) are more permissive to L. pneumophila legS2 mutants than wild-type (WT) strains. This permissiveness to L. pneumophila legS2 is neither attributed to abolished caspase-1, caspase-7 or caspase-3 activation, nor due to the impairment of phagosome-lysosome fusion. Instead, an infection with the legS2 mutant resulted in the reduction of some inflammatory cytokines and their corresponding mRNA; this effect is mediated by the inhibition of the nuclear transcription factor kappa-B (NF-κB). Moreover, BMDMs infected with L. pneumophila legS2 mutant showed elongated mitochondria that resembles mitochondrial fusion. Therefore, the absence of LegS2 effector is associated with reduced NF-κB activation and atypical morphology of mitochondria.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. L. pneumophila legS2 mutant replicates in human MDMs and wild-type mice and their derived macrophages.
A) BMDMs were infected with wild-type L. pneumophila, JR32, or ΔlegS2 for 1, 24, 48 and 72h. The colony forming units (CFUs) were scored at the indicated time points. B) hMDMs were infected with wild-type L. pneumophila, JR32, or ΔlegS2 for 1, 24, 48 and 72h. Data are shown as mean + SD of n = 3. Asterisks indicate significant differences, and a two tailed t-test was used to calculate the P value (*P<0.05, **P<0.01, ***P<0.001). (C and D) Four female C57BL/6 mice per group received 1x106 of JR32 or legs2 bacteria intratracheally. Lungs were homogenized and plated for CFUs, counting at (C) 4 or (D) 48 h post infection. (C and D) Data are shown as mean + SD of four mice. Asterisks indicate significant differences (***P<0.001); a two tailed t-test was used to calculate the P-value.
Fig 2
Fig 2. The permissiveness of mice BMDMs to legS2 mutant bacteria is independent of caspase-1 or caspase-7 activation.
(A) Caspase-1 KO (casp-1-/-) macrophages were infected with L. pneumophila, JR32, or legS2 with an MOI of 0.5 for 1, 24, 48 and 72 h; then, CFUs were measured at the indicated time points. (B) Levels of IL-1β were detected in supernatants of WT or casp-1-/- BMDMs were infected with JR32 or the legS2 mutant after 24 hr, while WT BMDMs were either not treated (NT) or infected with L. pneumophila, JR32, or legS2 mutant bacteria for 2 h. Salmonella infection (Sal) was used as a positive control for caspase-1 or caspase-7 activation. (C) Casp-1 or (D) casp-7 antibodies were used to detect casp-1 and casp-7 activation, respectively, in cell extracts.
Fig 3
Fig 3. Replication of legS2 mutant is independent of host cell death.
(A) Wild-type BMDMs were not treated (NT) or infected with the type IV secretion mutant dotA, wild-type L. pneumophila, JR32, or legS2 mutant for 24h at MOIs of 0.5 and 5. Then, the fold change in LDH release was measured from the overall population of macrophages. The data represents the mean + SD of n = 3. (B) Wild-type C57BL/6 (B6) were not treated (NT) or infected with wild-type L. pneumophila, JR32, legS2, or dotA mutant for 8 h. Salmonella infection (Sal) was used as a positive control for caspase-3 activation. A Western blot with caspase-3 antibody was used to detect casp-3 activation. β-actin was used as a loading control.
Fig 4
Fig 4. Replication of L. pneumophila legS2 mutant is not due to defective phagosome-lysosome fusion.
(A) Images of wild-type macrophages not infected (NT) or infected with the type IV secretion mutant dotA, wild-type L. pneumophila, JR32, and the legS2 mutant. The first panel presents staining with DAPI, with arrow heads pointing to the bacteria. The second panel shows staining with the lyso tracker. The third panel depicts merged images, with bacteria colocalized with the lysosomal marker. B) The percent of bacteria colocalized with the lyso tracker was scored in 100 infected cells from 3 independent coverslips. The data represents the mean + SD of n = 3.
Fig 5
Fig 5. Increased replication of the legS2 mutant in BMDMs is associated with reduced cytokine production.
WT BMDMs or casp-1-/- macrophages were infected with L. pneumophila, JR32, or the legS2 mutant. 24 h post infection, the supernatants from infected and un-infected cells were assayed by ELISA. (A) IL-10 level, (B) IL-6, (C) TNFα levels. Data represent the mean + SD of n = 3. Asterisks indicate significant differences (***P<0.001), and a two tailed t-test was used to calculate the P-value.
Fig 6
Fig 6. The legS2 mutant limits the levels of cytokine mRNAs.
WT BMDMs or casp-1-/- macrophages were infected with L. pneumophila, JR32, or the legS2 mutant. 4 h post infection, total RNA was isolated from JR32 or legS2 infected macrophages; then, the converted cDNA was used for quantitative PCR. The target gene Ct values were normalized to the Ct values of two housekeeping genes (human GAPDH and CAP-1, according to the cell origin) and expressed as relative copy numbers (RCN). (A) Represents the IL-10 RCNs, (B) Represents the IL-6 RCNs, (C) Represents the TNFα RCNs. Data represent the mean + SD of n = 3. Asterisks indicate significant differences (***P<0.001); a two tailed t-test was used to calculate the P-value.
Fig 7
Fig 7. The legS2 mutant inhibits the NF-κB pathway.
Wild-type macrophages were either not treated (NT) or infected with the JR32, or the legS2 mutant for 1, 4, and 8h. Then, nuclear extracts were processed using electrophoretic mobility shift assay (EMSA) to determine NF-κB activation.
Fig 8
Fig 8. Infection with the legS2 mutant is accompanied by change in mitochondrial morphology.
TEM of BMDMs infected with the JR32 or the legS2 mutant. Images were taken from 24 h post-infected cells.
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