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. 2016 Feb 16;113(7):1901-6.
doi: 10.1073/pnas.1522067113. Epub 2016 Feb 1.

Legionella pneumophila S1P-lyase _targets host sphingolipid metabolism and restrains autophagy

Monica Rolando  1 , Pedro Escoll  1 , Tamara Nora  1 , Joëlle Botti  2 , Valérie Boitez  2 , Carmen Bedia  3 , Craig Daniels  4 , Gilu Abraham  5 , Peter J Stogios  4 , Tatiana Skarina  4 , Charlotte Christophe  1 , Delphine Dervins-Ravault  1 , Christel Cazalet  1 , Hubert Hilbi  6 , Thusitha W T Rupasinghe  7 , Dedreia Tull  7 , Malcolm J McConville  8 , Sze Ying Ong  9 , Elizabeth L Hartland  9 , Patrice Codogno  2 , Thierry Levade  3 , Thomas Naderer  5 , Alexei Savchenko  4 , Carmen Buchrieser  10
Affiliations

Legionella pneumophila S1P-lyase _targets host sphingolipid metabolism and restrains autophagy

Monica Rolando et al. Proc Natl Acad Sci U S A. .

Abstract

Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to _target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

Keywords: Legionella pneumophila; autophagy; sphingolipids; sphingosine-1-phosphate lyase; virulence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Crystal structure of LpSPL compared with human SPL. (A) Domain architecture of LpSPL with a detailed depiction of the different domains (ER: endoplasmic reticulum; TM: transmembrane). (Inset) The two chains of the dimer. Dashes show residues that are missing due to absent or poor electron density. (B) Cartoon representation of the crystal structure of the LpSPL dimer (chain A and chain B). Colors and dashes are as in A. (C) Superposition of dimeric structures of LpSPL and human SPL (PDB code 4Q6R) colored by chain. The superposition of the full structure of LpSPL onto hSPL showed that the two proteins have a rmsd of 1.4 Å over 349 residues.
Fig. 2.
Fig. 2.
Characterization of conserved residues in active site and measurement of LpSpl enzymatic activity. (A) Putative active site of LpSpl colored according to Fig. 1A. (B) Active site of hSPL (9) colored according to Fig. 1A. The hSPL K353-PLP conjugate is shown in sticks. (C) SPL activity measured 48 h after transfection of HEK-293T cells with empty vectors (MOCK), LpSpl, or mSPL (mean ± SD; n = 3). (D) SPL activity measured 8 h after infection of MEF spl−/− with L. pneumophila with empty vector (wt), overexpressing LpSpl strain (wt+LpSpl), mutant strain with empty vector (Δspl), and complemented strain (Δspl+LpSpl) (mean ± SD; n = 3). Note that WT MEFs showed SPL activity of 138 pmol/h/mg. (E) SPL activity of wt or mutated LpSpl proteins (as indicated) in transfected HEK-293T cells (mean ± SD; n = 3).
Fig. 3.
Fig. 3.
LpSPL _targets host sphingosine to impair starvation-induced autophagy. (A) Relative levels of total lipids from THP-1 macrophages uninfected or infected with WT L. pneumophila or the Δspl mutant were extracted and analyzed by LC-MS (mean ± SD). (B) Levels of sphingosine were determined in uninfected THP-1 cells or infected with the WT or Δspl mutant carrying the empty vector (wt, Δspl) or the mutant expressing LpSpl (Δspl+LpSpl) (mean ± SD). (C) (Top) Immunoblot visualizing the LC3-I/LC3-II conversion in HEK-293T cells transfected for 48 h with empty vector (MOCK) or LpSpl kept in complete medium (CM) or minimum medium (EBSS) with or without BafA1. β-Actin was used as loading control. (Bottom) LC3II/Actin signal quantification (mean ± SD; n = 3). (D) Quantitative high-content analysis of GFP-LC3 puncta of cells transiently transfected with the empty vectors (MOCK), LpSPL, or mSPL expressing vectors in EBSS and BafA1. (Top) Representative pictures of GFP-LC3 puncta. (Scale bar, 10 µm.) (Bottom) Percentage of cells positive for GFP-LC3 puncta (mean ± SD; n = 3). (E) p62/Actin signal quantification of Western blot analysis of p62 degradation in HEK-293T cells transfected for 48 h with empty vectors (MOCK), mSPL, or LpSPL wild type (wt) or carrying single amino acid mutations, as indicated (mean ± SD; n = 3).
Fig. 4.
Fig. 4.
LpSpl limits autophagic response during L. pneumophila infection. (A) THP-1 cells were infected 2, 8, and 20 h with WT or Δspl strains (+DsRed vector). Quantitative high-content analysis of the percentage of total cells containing LC3 puncta are shown (>300 individual cells analyzed/condition) in the presence of BafA1 (mean ± SD; n = 3). (B) LC3 (green), nuclei (cyan), L. pneumophila (red), and F-actin (gray). Representative images of uninfected and 20-h infected cells in the same field. (Scale bar, 20 µm.) (C) Quantitative high-content analyses of the number of cells with LC3 puncta in the presence or not of BafA1 at 20 h postinfection (open circles: pooled single-well replicates; red line: mean ± SD). (D) Quantitative high-content analysis of LC3 puncta as in C; 8 h of infection with L. pneumophila strain Philadelphia wt, ΔravZ, Δspl, and Δspl/ravZ in the presence of BafA1. (E) Competitive infection of A/J mice measured after simultaneous intratracheal inoculation of ∼105 cfu of wt and Δspl mutant. Mice were killed 72 h following inoculation to examine the bacterial content of their lungs, and the competitive index was calculated (mean = 0.2588; P < 0.0001).
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