SummaryThe environmental pathogen Legionella pneumophila encodes three proteins containing F-box domains and additional protein-protein interaction domains, reminiscent of eukaryotic SCF ubiquitin-

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...

Although recombination during human immunodeficiency virus type 1 (HIV-1) replication in vitro and in vivo has been documented, little information is available concerning the extent that recombination contributes to the diversity of HIV-1 quasispecies in the course of infection in individual patents. To investigate the impact of recombination on viral diversity, we developed a technique that permits the isolation of contemporaneous clonal viral populations resulting from single infectious events by plasma-derived viruses, thereby permitting the assessment of recombination throughout the viral genomes, including widely separated loci, from individual patients. A comparison of the genomic sequences of clonal viruses from six patients, including patients failing treatment with antiretroviral therapy, demonstrated strong evidence for extensive recombination. Recombination increased viral diversity through two distinct mechanisms. First, evolutionary bottlenecks appeared to be restricted to minimal segments of the genome required to obtain selective advantage, thereby preserving diversity in adjacent regions. Second, recombination between adjacent gene segments appeared to generate diversity in both pol and env genes. Thus, the shuffling of resistance mutations within the genes coding for the protease and reverse transcriptase, as well as recombination between these regions, could increase the diversity of drug resistance genotypes. These findings demonstrate that recombination in HIV-1 contributes to the diversity of viral quasispecies by restricting evolutionary bottlenecks to gene segments and by generating novel genotypes in pol and env, supporting the idea that recombination may be critical to adaptive evolution of HIV in the face of constantly moving selective pressures, whether exerted by the immune system or antiretroviral therapy.