scholarly journals LtpD Is a Novel Legionella pneumophila Effector That Binds Phosphatidylinositol 3-Phosphate and Inositol Monophosphatase IMPA1

2013 ◽  
Vol 81 (11) ◽  
pp. 4261-4270 ◽  
Author(s):  
Clare R. Harding ◽  
Corinna Mattheis ◽  
Aurélie Mousnier ◽  
Clare V. Oates ◽  
Elizabeth L. Hartland ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Galleria Mellonella ◽  
Effector Proteins ◽  
Lung Epithelial Cells ◽  
Systematic Analysis ◽  
Content Type ◽  
Bacterial Replication ◽  
Phosphatidylinositol 3

ABSTRACTThe Dot/Icm type IV secretion system (T4SS) ofLegionella pneumophilais crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of theLegionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD471-626bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P]in vitroand colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae ofGalleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-bindingL. pneumophilaeffector that has a role in intracellular bacterial replication.

scholarly journals Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
A. Leoni Swart ◽  
Bernhard Steiner ◽  
Laura Gomez-Valero ◽  
Sabina Schütz ◽  
Mandy Hannemann ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Host Cells ◽  
Divergent Evolution ◽  
Ran Gtpase ◽  
Content Type ◽  
Gtpase Cycle ◽  
Gtpase Ran

ABSTRACT Legionella pneumophila governs its interactions with host cells by secreting >300 different “effector” proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG. The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975. legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection. IMPORTANCE Legionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires’ disease. The opportunistic pathogen grows in amoebae and macrophages by employing a “type IV” secretion system, which secretes more than 300 different “effector” proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.

scholarly journals Live-Cell Imaging of Phosphoinositide Dynamics and Membrane Architecture during Legionella Infection

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Stephen Weber ◽  
Maria Wagner ◽  
Hubert Hilbi
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Effector Proteins ◽  
Host Cells ◽  
Wild Type ◽  
Host Cell Membrane ◽  
Content Type ◽  
Temporal And Spatial

ABSTRACTThe causative agent of Legionnaires’ disease,Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, theLegionella-containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different “effector” proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoebaDictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficientL. pneumophila, PtdIns(3,4,5)P3transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3)Pwithin 1 min after uptake. Whereas phagosomes containing ΔicmTmutant bacteria remained decorated with PtdIns(3)P, more than 80% of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major rearrangement of PtdIns(3)P-positive membranes condensing to the cell center. PtdIns(4)Ptransiently localized to early phagosomes harboring wild-type or ΔicmT L. pneumophilaand was cleared within minutes after uptake. During the following 2 h, PtdIns(4)Psteadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)Pidentity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4)P-positive and ER membranes was even more pronounced for LCVs harboring ΔsidC-sdcAmutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions.IMPORTANCEThe environmental bacteriumLegionella pneumophilais the causative agent of Legionnaires’ pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, theLegionella-containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. UsingDictyosteliumamoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3)Pwas slowly cleared from LCVs, thus incapacitating the host cell’s digestive machinery, while PtdIns(4)Pgradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.

scholarly journals Noncanonical Inhibition of mTORC1 byCoxiella burnetiiPromotes Replication within a Phagolysosome-Like Vacuole

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Charles L. Larson ◽  
Kelsi M. Sandoz ◽  
Diane C. Cockrell ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Secretion System ◽  
Effector Proteins ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Content Type ◽  
Mechanistic Target Of Rapamycin ◽  
Infected Cells ◽  
Bacterial Replication

ABSTRACTThe Q fever agentCoxiella burnetiiis a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify theCoxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report thatC. burnetiiinhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However,C. burnetiidid not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 byC. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulatesC. burnetiiintracellular growth.IMPORTANCECoxiella burnetiiis an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of theCoxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report thatC. burnetiigrowth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefitC. burnetiigrowth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate thatC. burnetiiinhibition of mTORC1 without accelerated autophagy promotes bacterial growth.

scholarly journals Interaction of the Ankyrin H Core Effector of Legionella with the Host LARP7 Component of the 7SK snRNP Complex

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Juanita Von Dwingelo ◽  
Ivy Yeuk Wah Chung ◽  
Christopher T. Price ◽  
Lei Li ◽  
Snake Jones ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Host Protein ◽  
Host Cells ◽  
Transcriptional Elongation ◽  
Intracellular Growth ◽  
Pol Ii ◽  
Content Type ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT Species of the Legionella genus encode at least 18,000 effector proteins that are translocated through the Dot/Icm type IVB translocation system into macrophages and protist hosts to enable intracellular growth. Eight effectors, including ankyrin H (AnkH), are common to all Legionella species. The AnkH effector is also present in Coxiella and Rickettsiella. To date, no pathogenic effectors have ever been described that directly interfere with host cell transcription. We determined that the host nuclear protein La-related protein 7 (LARP7), which is a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, interacts with AnkH in the host cell nucleus. The AnkH-LARP7 interaction partially impedes interactions of the 7SK snRNP components with LARP7, interfering with transcriptional elongation by polymerase (Pol) II. Consistent with that, our data show AnkH-dependent global reprogramming of transcription of macrophages infected by Legionella pneumophila. The crystal structure of AnkH shows that it contains four N-terminal ankyrin repeats, followed by a cysteine protease-like domain and an α-helical C-terminal domain. A substitution within the β-hairpin loop of the third ankyrin repeat results in diminishment of LARP7-AnkH interactions and phenocopies the ankH null mutant defect in intracellular growth. LARP7 knockdown partially suppresses intracellular proliferation of wild-type (WT) bacteria and increases the severity of the defect of the ΔankH mutant, indicating a role for LARP7 in permissiveness of host cells to intracellular bacterial infection. We conclude that the AnkH-LARP7 interaction impedes interaction of LARP7 with 7SK snRNP, which would block transcriptional elongation by Pol II, leading to host global transcriptional reprogramming and permissiveness to L. pneumophila. IMPORTANCE For intracellular pathogens to thrive in host cells, an environment that supports survival and replication needs to be established. L. pneumophila accomplishes this through the activity of the ∼330 effector proteins that are injected into host cells during infection. Effector functions range from hijacking host trafficking pathways to altering host cell machinery, resulting in altered cell biology and innate immunity. One such pathway is the host protein synthesis pathway. Five L. pneumophila effectors have been identified that alter host cell translation, and 2 effectors have been identified that indirectly affect host cell transcription. No pathogenic effectors have been described that directly interfere with host cell transcription. Here we show a direct interaction of the AnkH effector with a host cell transcription complex involved in transcriptional elongation. We identify a novel process by which AnkH interferes with host transcriptional elongation through interference with formation of a functional complex and show that this interference is required for pathogen proliferation.

scholarly journals Lcl of Legionella pneumophila Is an Immunogenic GAG Binding Adhesin That Promotes Interactions with Lung Epithelial Cells and Plays a Crucial Role in Biofilm Formation

2011 ◽  
Vol 79 (6) ◽  
pp. 2168-2181 ◽  
Author(s):  
Carla Duncan ◽  
Akriti Prashar ◽  
Jannice So ◽  
Patrick Tang ◽  
Donald E. Low ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
Case Fatality ◽  
Lung Epithelial Cells ◽  
Molecular Evidence ◽  
Heparin Binding ◽  
Content Type ◽  
Lung Epithelial

ABSTRACTLegionellosis is mostly caused byLegionella pneumophilaand is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%.In vitroandin vivo, interactions ofL. pneumophilawith lung epithelial cells are mediated by the sulfated glycosaminoglycans (GAGs) of the host extracellular matrix. In this study, we have identified severalLegionellaheparin binding proteins. We have shown that one of these proteins, designated Lcl, is a polymorphic adhesin ofL. pneumophilathat is produced during legionellosis. Homologues of Lcl are ubiquitous inL. pneumophilaserogroups but are undetected in otherLegionellaspecies. Recombinant Lcl binds to GAGs, and a Δlpg2644mutant demonstrated reduced binding to GAGs and human lung epithelial cells. Importantly, we showed that the Δlpg2644strain is dramatically impaired in biofilm formation. These data delineate the role of Lcl in the GAG binding properties ofL. pneumophilaand provide molecular evidence regarding its role inL. pneumophilaadherence and biofilm formation.

scholarly journals Study ofLegionellaEffector Domains Revealed Novel and Prevalent Phosphatidylinositol 3-Phosphate Binding Domains

2019 ◽  
Vol 87 (6) ◽  
Author(s):  
Nimrod Nachmias ◽  
Tal Zusman ◽  
Gil Segal
Keyword(s):  
Legionella Pneumophila ◽  
Host Cells ◽  
Phosphate Binding ◽  
Content Type ◽  
Binding Domains ◽  
Type Iv ◽  
Charged Amino Acids ◽  
Overlay Assay ◽  
Phosphatidylinositol 3

ABSTRACTLegionella pneumophilaand otherLegionellaspecies replicate intracellularly using the Icm/Dot type IV secretion system. InL. pneumophilathis system translocates >300 effectors into host cells and in theLegionellagenus thousands of effectors were identified, the function of most of which is unknown. FourteenL. pneumophilaeffectors were previously shown to specifically bind phosphoinositides (PIs) using dedicated domains. We found that PI-binding domains of effectors are usually not homologous to one another; they are relatively small and located at the effectors' C termini. We used the previously identifiedLegionellaeffector domains (LEDs) with unknown function and the above characteristics of effector PI-binding domains to discover novel PI-binding LEDs. We identified three predicted PI-binding LEDs that are present in 14 L. pneumophilaeffectors and in >200 effectors in theLegionellagenus. Using anin vitroprotein-lipid overlay assay, we found that 11 of theseL. pneumophilaeffectors specifically bind phosphatidylinositol 3-phosphate (PI3P), almost doubling the number ofL. pneumophilaeffectors known to bind PIs. Further, we identified in each of these newly discovered PI3P-binding LEDs conserved, mainly positively charged, amino acids that are essential for PI3P binding. Our results indicate thatLegionellaeffectors harbor unique domains, shared by many effectors, which directly mediate PI3P binding.

scholarly journals In Vitro Characterization of Protein Effector Export in the Bradyzoite Stage of Toxoplasma gondii

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Joshua Mayoral ◽  
Peter Shamamian ◽  
Louis M. Weiss
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Persistent Infection ◽  
Chronic Infection ◽  
Life Stage ◽  
Effector Proteins ◽  
Content Type ◽  
Time Points ◽  
Bradyzoite Differentiation

ABSTRACT The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain chronic infection of its host for prolonged periods. Despite this, little is known regarding whether and how T. gondii bradyzoites, a quasi-dormant life stage residing within intracellular cysts, manipulate the host cell to maintain persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. Because MYR1 is known to be involved in the translocation of parasite-derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show, by immunofluorescence assays, that most effectors accumulate in the host nucleus at early but not late time points after infection, during the tachyzoite-to-bradyzoite transition and when parasites further along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon gamma signaling, which was previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications regarding how this highly successful parasite maintains persistent infection of its host. IMPORTANCE Toxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life stage successfully establishes and maintains persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different time points after bradyzoite differentiation and found that they accumulated largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediated its function after prolonged infection with bradyzoites, and we provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

scholarly journals Antimicrobial Activity of Solithromycin against Clinical Isolates of Legionella pneumophila Serogroup 1

2013 ◽  
Vol 58 (2) ◽  
pp. 909-915 ◽  
Author(s):  
Julia Mallegol ◽  
Prabhavathi Fernandes ◽  
Roberto G. Melano ◽  
Cyril Guyard
Keyword(s):  
Legionella Pneumophila ◽  
Lung Epithelial Cells ◽  
Clinical Isolates ◽  
Protein Synthesis Inhibitor ◽  
Respiratory Pathogens ◽  
Synthesis Inhibitor ◽  
Promising Alternative ◽  
Content Type ◽  
Broth Microdilution Method

ABSTRACTThe activity of solithromycin was evaluated against clinicalLegionella pneumophilaserogroup 1 (Lp1) isolates (n= 196) collected in Ontario, Canada, from 1980 to 2011. Itsin vitroactivity was compared to that of azithromycin (AZM) using the broth microdilution method. Solithromycin had a MIC50of ≤0.015 μg/ml and a MIC90of 0.031 μg/ml, making its activity at least 8-fold to 32-fold higher than that of AZM (MIC50and MIC90, 0.125 μg/ml and 1 μg/ml, respectively). Ninety-nine percent of the isolates had MICs for solithromycin ranging from ≤0.015 μg/ml to 0.031 μg/ml, whereas 83.6% of the isolates showed MICs for AZM ranging from 0.062 μg/ml to 0.25 μg/ml. Interestingly, 96.7% (30 out of 31 clinical isolates) identified with higher AZM MICs (0.5 μg/ml to 2 μg/ml) belonged to the clinically prevalent sequence type 1. To investigate the intracellular activity of solithromycin,in vitroinvasion assays were also performed against a subset of representative Lp1 isolates internalized within human lung epithelial cells. Solithromycin and AZM both inhibited growth of all intracellular Lp1 isolates at 1× or 8× MICs, displaying bacteriostatic effects, as would be expected with protein synthesis inhibitor rather than bactericidal activity. Solithromycin demonstrated the highestin vitroand intracellular potency against all Lp1 isolates compared to AZM. Given the rapid spread of resistance mechanisms among respiratory pathogens and the reported treatment failures in legionellosis, the development of this new fluoroketolide, already in phase 3 oral clinical studies, constitutes a promising alternative option for the treatment of legionellosis.

scholarly journals Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila

2016 ◽  
Vol 84 (8) ◽  
pp. 2185-2197 ◽  
Author(s):  
Tamara J. O'Connor ◽  
Huaixin Zheng ◽  
Susan M. VanRheenen ◽  
Soma Ghosh ◽  
Nicholas P. Cianciotto ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Iron Acquisition ◽  
Dominant Role ◽  
Bacterial Pathogen ◽  
Transport Protein ◽  
Iron Limitation ◽  
Growth Defect ◽  
Content Type

Legionella pneumophilais an intracellular bacterial pathogen that replicates in alveolar macrophages, causing a severe form of pneumonia. Intracellular growth of the bacterium depends on its ability to sequester iron from the host cell. In theL. pneumophilastrain 130b, one mechanism used to acquire this essential nutrient is the siderophore legiobactin. Iron-bound legiobactin is imported by the transport protein LbtU. Here, we describe the role of LbtP, a paralog of LbtU, in iron acquisition in theL. pneumophilastrain Philadelphia-1. Similar to LbtU, LbtP is a siderophore transport protein and is required for robust growth under iron-limiting conditions. Despite their similar functions, however, LbtU and LbtP do not contribute equally to iron acquisition. The Philadelphia-1 strain lacking LbtP is more sensitive to iron deprivationin vitro. Moreover, LbtP is important forL. pneumophilagrowth within macrophages while LbtU is dispensable. These results demonstrate that LbtP plays a dominant role over LbtU in iron acquisition. In contrast, loss of both LbtP and LbtU does not impairL. pneumophilagrowth in the amoebal hostAcanthamoeba castellanii, demonstrating a host-specific requirement for the activities of these two transporters in iron acquisition. The growth defect of the ΔlbtPmutant in macrophages is not due to alterations in growth kinetics. Instead, the absence of LbtP limitsL. pneumophilareplication and causes bacteria to prematurely exit the host cell. These results demonstrate the existence of a preprogrammed exit strategy in response to iron limitation that allowsL. pneumophilato abandon the host cell when nutrients are exhausted.

scholarly journals Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor

2015 ◽  
Vol 83 (10) ◽  
pp. 3989-4002 ◽  
Author(s):  
Gunnar N. Schroeder ◽  
Philipp Aurass ◽  
Clare V. Oates ◽  
Edward W. Tate ◽  
Elizabeth L. Hartland ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Virulence Factor ◽  
Phospholipase D ◽  
Membrane Trafficking ◽  
Legionella Pneumophila ◽  
Cultured Cells ◽  
Effector Proteins ◽  
Subcellular Targeting ◽  
Content Type

Legionella pneumophilais a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence ofL. pneumophiladepends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, theLegionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA)in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, anL. pneumophilaΔlpdAmutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing toL. pneumophilainfectionin vivo.

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