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1.
mBio ; 14(5): e0094323, 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-37676004

RESUMO

IMPORTANCE: Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.


Assuntos
Dictyostelium , Mycobacterium marinum , Mycobacterium tuberculosis , Tuberculose , Humanos , Vacúolos/metabolismo , Dictyostelium/microbiologia , Retículo Endoplasmático , Mycobacterium marinum/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/metabolismo
2.
Elife ; 122023 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-37158597

RESUMO

The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.


Assuntos
Dictyostelium , Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Legionella pneumophila/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Macrófagos/metabolismo , Dictyostelium/metabolismo , Gotículas Lipídicas/metabolismo , Vacúolos/metabolismo , Legionella/metabolismo , Doença dos Legionários/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
3.
Microlife ; 4: uqad018, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37223745

RESUMO

Vesicular trafficking and membrane fusion are well-characterized, versatile, and sophisticated means of 'long range' intracellular protein and lipid delivery. Membrane contact sites (MCS) have been studied in far less detail, but are crucial for 'short range' (10-30 nm) communication between organelles, as well as between pathogen vacuoles and organelles. MCS are specialized in the non-vesicular trafficking of small molecules such as calcium and lipids. Pivotal MCS components important for lipid transfer are the VAP receptor/tether protein, oxysterol binding proteins (OSBPs), the ceramide transport protein CERT, the phosphoinositide phosphatase Sac1, and the lipid phosphatidylinositol 4-phosphate (PtdIns(4)P). In this review, we discuss how these MCS components are subverted by bacterial pathogens and their secreted effector proteins to promote intracellular survival and replication.

4.
EMBO Rep ; 24(3): e56007, 2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36588479

RESUMO

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.


Assuntos
Dictyostelium , Legionella pneumophila , Legionella , Vacúolos/metabolismo , Legionella/metabolismo , Dictyostelium/microbiologia , Fosfatidilinositóis/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/metabolismo
5.
EMBO Rep ; 22(9): e52972, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34314090

RESUMO

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.


Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Proteínas de Bactérias/genética , Humanos , Legionella/genética , Legionella pneumophila/genética , Percepção de Quorum , Vacúolos
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