| 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.The 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, SPL homologs have now been identified in other bacteria such as Roseiflexus, Myxococcus, Stigmatella, and Symbiobacterium (6).Eukaryotic SPL tightly regulates intracellular levels of sphingosine-1-phosphate (S1P). Sphingolipids are ubiquitous building blocks of eukaryotic cell membranes, and the sphingolipid metabolites ceramide, ceramide-1-phosphate, sphingosine, and S1P are key signaling molecules that regulate many cellular processes important in immunity, inflammation, infection, and cancer (7). SPL uses pyridoxal 5′-phosphate (PLP) as a cofactor to irreversibly degrade S1P into phosphoethanolamine and hexadecenal (SI Appendix, Fig. S1). Structural analysis of SPL from Symbiobacterium thermophilum (StSPL) and Saccharomyces cerevisiae (Dpl1p) identified the residues involved in activity and proposed a mechanism for S1P cleavage (8). Structural elucidation of human SPL (hSPL) showed that the yeast and the human enzymes adopt largely the same structures (9).Recent work suggests a possible link between the role of lipids in the regulation of apoptosis and autophagy (10). Autophagy is an evolutionary conserved pathway controlling the quality and quantity of eukaryotic organelles and the cytoplasmic biomass. Double-membrane vesicles called “autophagosomes” engulf nonfunctional or damaged cellular components and deliver them to lysosomes, where the content is degraded (11). Furthermore, it has been shown that autophagy acts as a cell-autonomous defense mechanism against intracellular bacteria, contributing to antibacterial immunity by regulating the inflammatory immune response and routing engulfed intracellular bacteria toward lysosomal degradation (12, 13). Many pathogens are able to evade autophagy, although the molecular mechanisms at play remain largely uncharacterized (14–20). Among these pathogens L. pneumophila is known to escape cellular attack by blocking autophagy defenses (21). Although it has been reported that L. pneumophila interferes with the autophagy machinery and with host factors that play a role in the cellular defense (22), only two L. pneumophila proteins that target the autophagy machinery, RavZ and LegA9, have been identified (23, 24). The bacterial effector RavZ is a cysteine protease that cleaves and causes delipidation of the autophagosome protein LC3, thereby dampening the autophagy process (23, 25). Interestingly, RavZ is not present in all strains of L. pneumophila, but in all strains tested, L. pneumophila-mediated disruption of autophagosomal maturation delays and/or prevents the Legionella-containing vacuole (LCV) from fusing with lysosomes (26). Thus, L. pneumophila likely also employs other mechanisms to restrain autophagy, and LpSpl, present in all strains sequenced to date, is a good candidate. A first characterization of LpSpl in strain JR32 showed that this protein is secreted by the Dot/Icm type IV secretion system and that it complements the sphingosine-sensitive phenotype of a S. cerevisiae SPL-null mutant, suggesting that it indeed has SPL activity. However, no functional analyses were reported (5). Thus, we aimed to understand the function of this SPL homolog in L. pneumophila.Here we report the crystal structure of LpSpl, identify the active site, and show that LpSpl indeed confers S1P lyase activity to L. pneumophila. Its activity during infection leads to the specific reduction of cellular levels of sphingosine, whereas other sphingolipids are down-regulated in a LpSpl-independent manner. Furthermore, we confirm that LpSpl plays a role in delaying the autophagy response of the cell during infection and is important for infection in a pulmonary mouse model of Legionnaires’ disease. |
