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

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.

Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses.

BACKGROUND: Numerous studies have shown that viral quasi-species with genetically diverse envelope proteins (Env) replicate simultaneously in patients infected with the human immunodeficiency virus type 1 (HIV-1). Less information is available concerning the extent that envelope sequence diversity translates into a diversity of phenotypic properties, including infectivity and resistance to entry inhibitors. METHODS: To study these questions, we isolated genetically distinct contemporaneous clonal viral populations from the plasma of 5 HIV-1 infected individuals (n = 70), and evaluated the infectivity of recombinant viruses expressing Env proteins from the clonal viruses in several target cells. The sensitivity to entry inhibitors (enfuvirtide, TAK-799), soluble CD4 and monoclonal antibodies (2G12, 48d, 2F5) was also evaluated for a subset of the recombinant viruses (n = 20). RESULTS: Even when comparisons were restricted to viruses with similar tropism, the infectivity for a given target cell of viruses carrying different Env proteins from the same patient varied over an approximately 10-fold range, and differences in their relative ability to infect different target cells were also observed. Variable region haplotypes associated with high and low infectivity could be identified for one patient. In addition, clones carrying unique mutations in V3 often displayed low infectivity. No correlation was observed between viral infectivity and sensitivity to inhibition by any of the six entry inhibitors evaluated, indicating that these properties can be dissociated. Significant inter-patient differences, independent of infectivity, were observed for the sensitivity of Env proteins to several entry inhibitors and their ability to infect different target cells. CONCLUSION: These findings demonstrate the marked functional heterogeneity of HIV-1 Env proteins expressed by contemporaneous circulating viruses, and underscore the advantage of clonal analyses in characterizing the spectrum of functional properties of the genetically diverse viral populations present in a given patient.

Molecular mimicry: an important virulence strategy employed by Legionella pneumophila to subvert host functions.

It is 32 years since Legionella pneumophila was identified and recognized as a human pathogen, causing the severe form of pneumonia termed Legionnaires' disease, or legionellosis. This bacterium is found in freshwater reservoirs where it replicates in aquatic protozoa and can invade man-made water distribution systems. Although the disease can be treated by antibiotherapy and prevented through surveillance and control measures, reported cases of Legionnaires' disease continue to rise across Europe and outbreaks of major public health significance still occur. Genome sequencing and analyses led to a giant step forward by suggesting new ways by which this intracellular bacterium might subvert host functions. One particular feature revealed was the presence of many eukaryotic-like proteins, possibly mimicking host proteins to allow intracellular replication of Legionella. Here, we describe the identification and analysis of these proteins and report on recent advances detailing the mechanisms by which these proteins function. Finally, comparative and evolutionary genomic aspects regarding the eukaryotic-like proteins are presented. Collectively, these data have shed new light on the virulence strategies of L. pneumophila, a major aspect of which is molecular mimicry.

Contribution of recombination to the evolution of human immunodeficiency viruses expressing resistance to antiretroviral treatment.

Viral recombination has been postulated to play two roles in the development of human immunodeficiency virus (HIV) resistance to antiretroviral drugs. First, recombination has the capacity to associate resistance mutations expressed by distinct viruses, thereby contributing to the development of viruses with improved drug resistance. In addition, recombination could preserve diversity in regions outside those subject to strong selective pressure. In this study, we sought direct evidence for the occurrence of these processes in vivo by evaluating clonal virus populations obtained from the same patient before and after a treatment change that, while unsuccessful in controlling viral replication, led to the emergence of viruses expressing a different profile of resistance mutations. Phylogenetic studies supported the conclusion that the genotype arising after the treatment change resulted from the emergence of recombinant viruses carrying previously existing resistance mutations in novel combinations, whereas alternative explanations, including convergent evolution, were not consistent with observed genotypic changes. Despite evidence for a strong loss of genetic diversity in genomic regions coding for the protease and reverse transcriptase, diversity in regions coding for Gag and envelope was considerably higher, and recombination between the emerging viruses expressing the new pattern of resistance mutations and viral quasispecies in the previously dominant population contributed to this preservation of diversity in the envelope gene. These findings emphasize that recombination can participate in the adaptation of HIV to changing selective pressure, both by generating novel combinations of resistance mutations and by maintaining diversity in genomic regions outside those implicated in a selective sweep.

Extensive recombination among human immunodeficiency virus type 1 quasispecies makes an important contribution to viral diversity in individual patients.

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.