Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling.

Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purified LCVs indicate the presence of membrane contact sites (MCS) proteins possibly implicated in lipid exchange. Using dually fluorescence-labeled Dictyostelium discoideum amoeba, we reveal that VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L. pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L. pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.

The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote interbacterial and interkingdom signaling.

Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires' disease. The opportunistic pathogen employs the α-hydroxy-ketone compound Legionella autoinducer-1 (LAI-1) for intraspecies and interkingdom signaling. LAI-1 is produced by the autoinducer synthase Legionella quorum sensing A (LqsA), but it is not known, how LAI-1 is released by the pathogen. Here, we use a Vibrio cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in Escherichia coli generated LAI-1, which partitions to outer membrane vesicles (OMVs) and increases OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L.pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favor of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes interbacterial communication and interactions with eukaryotic host cells.

Tapping lipid droplets: A rich fat diet of intracellular bacterial pathogens.

Lipid droplets (LDs) are dynamic and versatile organelles present in most eukaryotic cells. LDs consist of a hydrophobic core of neutral lipids, a phospholipid monolayer coat, and a variety of associated proteins. LDs are formed at the endoplasmic reticulum and have diverse roles in lipid storage, energy metabolism, membrane trafficking, and cellular signaling. In addition to their physiological cellular functions, LDs have been implicated in the pathogenesis of several diseases, including metabolic disorders, cancer, and infections. A number of intracellular bacterial pathogens modulate and/or interact with LDs during host cell infection. Members of the genera Mycobacterium, Legionella, Coxiella, Chlamydia, and Salmonella exploit LDs as a source of intracellular nutrients and membrane components to establish their distinct intracellular replicative niches. In this review, we focus on the biogenesis, interactions, and functions of LDs, as well as on their role in lipid metabolism of intracellular bacterial pathogens.

Systematic exploration of Escherichia coli phage-host interactions with the BASEL phage collection.

Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of the molecular mechanisms underlying phage-host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage-host interactions by composing a well-assorted library of 68 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL (BActeriophage SElection for your Laboratory) collection. This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside 10 well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to 11 defense systems across different layers of bacterial immunity, and matched these results to the phages' host range across a panel of pathogenic enterobacterial strains. Clear patterns in the distribution of phage phenotypes and genomic features highlighted systematic differences in the potency of different immunity systems and suggested the molecular basis of receptor specificity in several phage groups. Our results also indicate strong trade-offs between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific ecological niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications.

Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery.

The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella-containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non-virulent and a non-replicating, virulent/transmissive phase. Here, we show on a single-cell level that at late stages of infection, individual motile (PflaA -GFP-positive) and virulent (PralF - and PsidC -GFP-positive) L. pneumophila emerge in the cluster of non-growing bacteria within an LCV. Comparative proteomics of PflaA -GFP-positive and PflaA -GFP-negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the PflaA -GFP-positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi-phasic life cycle of L. pneumophila.

Migration of Acanthamoeba through Legionella biofilms is regulated by the bacterial Lqs-LvbR network, effector proteins and the flagellum.

The environmental bacterium Legionella pneumophila causes the pneumonia Legionnaires' disease. The opportunistic pathogen forms biofilms and employs the Icm/Dot type IV secretion system (T4SS) to replicate in amoebae and macrophages. A regulatory network comprising the Legionella quorum sensing (Lqs) system and the transcription factor LvbR controls bacterial motility, virulence and biofilm architecture. Here we show by comparative proteomics that in biofilms formed by the L. pneumophila ΔlqsR or ΔlvbR regulatory mutants the abundance of proteins encoded by a genomic 'fitness island', metabolic enzymes, effector proteins and flagellar components (e.g. FlaA) varies. ∆lqsR or ∆flaA mutants form 'patchy' biofilms like the parental strain JR32, while ∆lvbR forms a 'mat-like' biofilm. Acanthamoeba castellanii amoebae migrated more slowly through biofilms of L. pneumophila lacking lqsR, lvbR, flaA, a functional Icm/Dot T4SS (∆icmT), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ∆lvbR or ∆icmT biofilms but not in ∆lqsR or ∆flaA biofilms. The amoeba-adherent bacteria induced promoters implicated in motility (PflaA ) or virulence (PsidC , PralF ). Taken together, the Lqs-LvbR network (quorum sensing), FlaA (motility) and the Icm/Dot T4SS (virulence) regulate migration of A. castellanii through L. pneumophila biofilms, and - apart from the T4SS - govern bacterial cluster formation on the amoebae.

Microbe Profile: Legionella pneumophila - a copycat eukaryote.

Legionella pneumophila is an environmental bacterium that parasitizes aquatic protozoa and uses the same processes to infect humans. The facultative intracellular pathogen causes a life-threatening pneumonia with possible systemic complications. The co-evolution with protozoa is reflected in an armoury of bacterial effectors, and many of these type IV-secreted proteins have likely been acquired by interdomain horizontal gene transfer (HGT) from hosts. The unique features of L. pneumophila are the largest bacterial effector repertoire known to date, subversion of virtually all eukaryotic signalling pathways and acquisition of eukaryotic enzyme activities used to manipulate the host cell to the pathogen's advantage.

The Legionella Lqs-LvbR Regulatory Network Controls Temperature-Dependent Growth Onset and Bacterial Cell Density.

Legionella species are facultative intracellular pathogens that cause a life-threatening pneumonia termed Legionnaires' disease. Legionella pneumophila employs the Lqs-LvbR (Legionella quorum sensing-Legionella virulence and biofilm regulator) network to regulate virulence and motility, but its role for growth in media is ill-defined. Here, we report that compared to the L. pneumophila reference strain JR32, a ΔlqsR mutant showed a reduced lag phase at 30°C and reached a higher cell density at 45°C, while the ΔlqsA, ΔlqsS, and ΔlqsT mutants showed a longer lag phase and reached a lower cell density. A ΔlvbR mutant resumed growth like the parental strain at 30°C but exhibited a substantially reduced cell density at 45°C. Thus, LvbR is an important cell density regulator at elevated temperatures. Environmental and clinical L. pneumophila strains grew in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) medium after distinct lag phases with similar rates at 30°C, reached different cell densities at the optimal growth temperature of 40°C, and no longer grew at 50°C. Legionella longbeachae reached a rather low cell density at 40°C and did not grow at and beyond 45°C. Genes encoding components of the Lqs-LvbR network were present in the genomes of the environmental and clinical L. pneumophila isolates, and upon growth at 30°C or 45°C, the PlqsR, PlqsA, PlqsS, and PlvbR promoters from strain JR32 were expressed in these strains with distinct patterns. Taken together, our results indicate that the Lqs-LvbR network governs the temperature-dependent growth onset and cell density of the L. pneumophila reference strain JR32 and possibly also of environmental and clinical L. pneumophila isolates. IMPORTANCE Environmental bacteria of the genus Legionella are the causative agents of the severe pneumonia Legionnaires' disease, the incidence of which is on the rise worldwide. Legionella pneumophila and Legionella longbeachae are the clinically most relevant species. The opportunistic pathogens are inhaled through contaminated aerosols and replicate in human lung macrophages with a mechanism similar to that in their natural hosts, free-living amoebae. Given their prevalence in natural and technical water systems, an efficient control of Legionella spp. by physical, chemical, or biological means will reduce the incidence of Legionnaires' disease. Here, we show that the Legionella quorum sensing (Lqs) system and the pleiotropic transcription factor LvbR govern the temperature-dependent growth onset and cell density of bacterial cultures. Hence, the growth of L. pneumophila in water systems is determined not only by the temperature and nutrient availability but also by quorum sensing, i.e., density- and signaling molecule-dependent gene regulation.

Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole.

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.

The structure of the Legionella response regulator LqsR reveals amino acids critical for phosphorylation and dimerization.

The water-borne bacterium Legionella pneumophila replicates in environmental protozoa and upon inhalation destroys alveolar macrophages, thus causing a potentially fatal pneumonia termed 'Legionnaires' disease'. L. pneumophila employs the Legionella quorum sensing (Lqs) system to control its life cycle, pathogen-host cell interactions, motility and natural competence. Signaling through the Lqs system occurs through the α-hydroxyketone compound Legionella autoinducer-1 (LAI-1) and converges on the prototypic response regulator LqsR, which dimerizes upon phosphorylation of the conserved aspartate, D108 . In this study, we determine the high-resolution structure of monomeric LqsR. The structure reveals a receiver domain adopting a canonical (ßα)5 fold, which is connected through an additional sixth helix and an extended α5-helix to a novel output domain. The two domains delineate a mainly positively charged groove, and the output domain adopts a five-stranded antiparallel ß-sheet fold similar to nucleotide-binding proteins. Structure-based mutagenesis identified amino acids critical for LqsR phosphorylation and dimerization. Upon phosphorylation, the LqsRD172A and LqsRD302N/E303Q mutant proteins dimerized even more readily than wild-type LqsR, and no evidence for semi-phosphorylated heterodimers was obtained. Taken together, the high-resolution structure of LqsR reveals functionally relevant amino acid residues implicated in signal transduction of the prototypic response regulator.

PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.

Evolution and function of bacterial RCC1 repeat effectors.

Intracellular bacterial pathogens harbour genes, the closest homologues of which are found in eukaryotes. Regulator of chromosome condensation 1 (RCC1) repeat proteins are phylogenetically widespread and implicated in protein-protein interactions, such as the activation of the small GTPase Ran by its cognate guanine nucleotide exchange factor, RCC1. Legionella pneumophila and Coxiella burnetii, the causative agents of Legionnaires' disease and Q fever, respectively, harbour RCC1 repeat coding genes. Legionella pneumophila secretes the RCC1 repeat 'effector' proteins LegG1, PpgA and PieG into eukaryotic host cells, where they promote the activation of the pleiotropic small GTPase Ran, microtubule stabilisation, pathogen vacuole motility and intracellular bacterial growth as well as host cell migration. The RCC1 repeat effectors localise to the pathogen vacuole or the host plasma membrane and target distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself. Coxiella burnetii translocates the RCC1 repeat effector NopA into host cells, where the protein localises to nucleoli. NopA binds to Ran GTPase and promotes the nuclear accumulation of Ran(GTP), thus pertubing the import of the transcription factor NF-κB and innate immune signalling. Hence, divergent evolution of bacterial RCC1 repeat effectors defines the range of Ran GTPase cycle targets and likely allows fine-tuning of Ran GTPase activation by the pathogens at different cellular sites.

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes.

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium-containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid-bound mycobactin (MBT) and the water-soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe-MBT or Fe-cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe-cMBT promoted the growth of wild-type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild-type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra- and intracellular growth of the pathogen.

Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol.

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication-permissive compartment termed the Mycobacterium-containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid-borne genes. Fluorescence microscopy of M. marinum-infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol-3-phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V-ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.

The pleiotropic Legionella transcription factor LvbR links the Lqs and c-di-GMP regulatory networks to control biofilm architecture and virulence.

The causative agent of Legionnaires' disease, Legionella pneumophila, colonizes amoebae and biofilms in the environment. The opportunistic pathogen employs the Lqs (Legionella quorum sensing) system and the signalling molecule LAI-1 (Legionella autoinducer-1) to regulate virulence, motility, natural competence and expression of a 133 kb genomic "fitness island", including a putative novel regulator. Here, we show that the regulator termed LvbR is an LqsS-regulated transcription factor that binds to the promoter of lpg1056/hnox1 (encoding an inhibitor of the diguanylate cyclase Lpg1057), and thus, regulates proteins involved in c-di-GMP metabolism. LvbR determines biofilm architecture, since L. pneumophila lacking lvbR accumulates less sessile biomass and forms homogeneous mat-like structures, while the parental strain develops more compact bacterial aggregates. Comparative transcriptomics of sessile and planktonic ΔlvbR or ΔlqsR mutant strains revealed concerted (virulence, fitness island, metabolism) and reciprocally (motility) regulated genes in biofilm and broth respectively. Moreover, ΔlvbR is hyper-competent for DNA uptake, defective for phagocyte infection, outcompeted by the parental strain in amoebae co-infections and impaired for cell migration inhibition. Taken together, our results indicate that L. pneumophila LvbR is a novel pleiotropic transcription factor, which links the Lqs and c-di-GMP regulatory networks to control biofilm architecture and pathogen-host cell interactions.

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila.

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER-associated compartment termed the Legionella-containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule-resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant-negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P-positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1-dependent aggregation of purified, ER-positive LCVs in vitro Thus, Sey1/Atl3-dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.

Comparative Proteomics of Purified Pathogen Vacuoles Correlates Intracellular Replication of Legionella pneumophila with the Small GTPase Ras-related protein 1 (Rap1).

Legionella pneumophila is an opportunistic bacterial pathogen that causes a severe lung infection termed "Legionnaires' disease." The pathogen replicates in environmental protozoa as well as in macrophages within a unique membrane-bound compartment, the Legionella-containing-vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates ca. 300 "effector proteins" into host cells, where they target distinct host factors. The L. pneumophila "pentuple" mutant (Δpentuple) lacks 5 gene clusters (31% of the effector proteins) and replicates in macrophages but not in Dictyostelium discoideum amoeba. To elucidate the host factors defining a replication-permissive compartment, we compare here the proteomes of intact LCVs isolated from D. discoideum or macrophages infected with Δpentuple or the parental strain Lp02. This analysis revealed that the majority of host proteins are shared in D. discoideum or macrophage LCVs containing the mutant or the parental strain, respectively, whereas some proteins preferentially localize to distinct LCVs. The small GTPase Rap1 was identified on D. discoideum LCVs containing strain Lp02 but not the Δpentuple mutant and on macrophage LCVs containing either strain. The localization pattern of active Rap1 on D. discoideum or macrophage LCVs was confirmed by fluorescence microscopy and imaging flow cytometry, and the depletion of Rap1 by RNA interference significantly reduced the intracellular growth of L. pneumophila Thus, comparative proteomics identified Rap1 as a novel LCV host component implicated in intracellular replication of L. pneumophila.

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.

IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid.

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Formation of the Legionella-containing vacuole: phosphoinositide conversion, GTPase modulation and ER dynamics.

The environmental bacterium Legionella pneumophila replicates in free-living amoeba as well as in alveolar macrophages upon inhalation of bacteria-laden aerosols. Resistance of the opportunistic pathogen to macrophages is a prerequisite to cause a severe pneumonia called Legionnaires' disease. L. pneumophila grows intracellularly in a unique, ER-associated compartment, the Legionella-containing vacuole (LCV). The bacterial Icm/Dot type IV secretion system represents an essential virulence factor, which translocates approximately 300 "effector proteins" into protozoan or mammalian host cells. Some of these effectors contribute to the formation of the LCV by targeting conserved host factors implicated in membrane dynamics, such as phosphoinositide lipids and small GTPases. Here we review recent findings on the role of phosphoinositides, small and large GTPases as well as ER dynamics for pathogen vacuole formation and intracellular replication of L. pneumophila.