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

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.Legionella pneumophila | sphingosine-1-phosphate lyase | autophagy | sphingolipids | virulence T he Gram-negative intracellular bacterium Legionella pneumophila is an opportunistic human pathogen responsible for Legionnaires' disease. The bacteria are naturally found in freshwater systems where they replicate within protozoan hosts (1). It is thought that the adaptation to replication within amoebas has equipped L. pneumophila with the factors required to replicate successfully within human macrophages following opportunistic infection (2). Through genome sequencing, we have discovered that L. pneumophila encodes a high number and variety of proteins similar in sequence to eukaryotic proteins that are never or rarely found in other prokaryotic genomes (3). Subsequent phylogenetic analyses have suggested that many of these proteins were acquired by horizontal gene transfer (3, 4). One of these proteins exhibits a high degree of similarity to eukaryotic sphingosine-1 phosphate lyase (SPL). The L. pneumophila SPL homolog (LpSpl encoded by gene lpp2128, lpg2176, or legS2) is conserved in all L. pneumophila strains sequenced to date, but absent from Legionella longbeachae (SI Appendix, Table S1). Phylogenetic analysis of SPL sequences showed that the L. pneumophila spl gene was most likely acquired early during evolution by horizontal gene transfer from a protozoan organism (4, 5). With the increase in genome sequences available...

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

“…A well-characterized example is Legionella pneumophila , which possesses the effector RavZ to catalyze the irreversible uncoupling of Atg8-family proteins from phosphatidylethnolamine on autophagosonal membranes, allowing L. pneumophila to inhibit autophagy [36,37]. Additionally, the effector Lpg1137 has recently been reported to proteolytically cleave STX17, a protein involved in the fusion of autophagosomes with the lysosome, and a further effector LpSpI, inhibits autophagy through its ability to target host sphingosine biosynthesis [38,39]. In contrast, rather than being bactericidal, the lysosome provides the appropriate environment to induce metabolic activity of Coxiella and promote intracellular replication [8].…”

Bacterial Pathogens versus Autophagy: Implications for Therapeutic Interventions

“…Legionella pneumophila survives within a vacuole that is targeted by LC3 during infection in macrophages, but blocks acidification by cleaving membrane-conjugated LC3 via the effector protein RavZ[53]. In addition, L. pneumophila blocks autophagy during infection of macrophages by translocating the effector protein sphingosine-1 phosphate lyase (LpSpl), and this triggers a reduction of several host sphingolipids critical for autophagy[54]. Serratia marcescens replicates and persists inside large membrane-bound compartments in epithelial cells that colocalize with LC3 but are non-acidic, indicating that the bacteria prevent fusion of the vesicles with lysosomes to generate a suitable niche for proliferation inside the host cell[55].…”

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei

“…Although the crystal structures of S1PLs from S. thermophilum , S. cerevisiae , humans, and L. pneumophila have been solved, the exact nature of substrate binding and enzyme catalysis are still unclear because the structure of a S1P-bound complex has not been determined (11, 18, 44). For example, in the putative mechanism, the active site base that deprotonates the S1P hydroxyl leading to the retro-aldol-like cleavage of the substrate has not been identified (supplemental Fig.…”

“…Recent studies have begun to address these questions. A bacterial S1PL homolog (LegS2) was identified in the intracellular human pathogen, L. pneumophila (17, 18), and its role as a virulence factor in the pathogenic mechanism of this organism was explored. B. pseudomallei is an intracellular Gram-negative pathogen that causes serious disease in humans and animals and, as such, is classified by the Centre for Disease Control as a category B biothreat agent.…”