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Cell-selective proteomics reveal novel effectors secreted by an obligate intracellular bacterial pathogen

ABSTRACT: Pathogenic bacteria secrete protein effectors to hijack host machinery and remodel their infectious niche. Rickettsia spp. are obligate intracellular bacteria that can cause life-threatening disease, but their absolute dependence on the host cell environment has impeded discovery of rickettsial effectors and their host targets. We implemented bioorthogonal non-canonical amino acid tagging (BONCAT) during R. parkeri infection to selectively label, isolate, and identify secreted effectors. As the first use of BONCAT in an obligate intracellular bacterium, our screen more than doubles the number of experimentally validated effectors for R. parkeri. The novel secreted rickettsial factors (Srfs) we identified include Rickettsia-specific proteins of unknown function that localize to the host cytoplasm, mitochondria, and ER. We further show that one such effector, SrfD, interacts with the host Sec61 translocon. Altogether, our work uncovers a diverse set of previously uncharacterized rickettsial effectors and lays the foundation for a deeper exploration of the host-pathogen interface.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Rickettsia Parkeri Str. Portsmouth (ncbitaxon:1105108)

SUBMITTER: Rebecca Lamason

PROVIDER: MSV000093380 | MassIVE | Mon Nov 13 07:28:00 GMT 2023

REPOSITORIES: MassIVE

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Cell-selective proteomics reveal novel effectors secreted by an obligate intracellular bacterial pathogen

Project description:Pathogenic bacteria secrete protein effectors to hijack host machinery and remodel their infectious niche. Rickettsia spp. are obligate intracellular bacteria that can cause life-threatening disease, but their absolute dependence on the host cell environment has impeded discovery of rickettsial effectors and their host targets. We implemented bioorthogonal non-canonical amino acid tagging (BONCAT) during R. parkeri infection to selectively label, isolate, and identify secreted effectors. As the first use of BONCAT in an obligate intracellular bacterium, our screen more than doubles the number of experimentally validated effectors for R. parkeri. The novel secreted rickettsial factors (Srfs) we identified include Rickettsia-specific proteins of unknown function that localize to the host cytoplasm, mitochondria, and ER. We further show that one such effector, SrfD, interacts with the host Sec61 translocon. Altogether, our work uncovers a diverse set of previously uncharacterized rickettsial effectors and lays the foundation for a deeper exploration of the host-pathogen interface.

2023-11-13 | MSV000093381 | MassIVE

The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state

Project description:Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium in the family Rickettsiaceae that causes scrub typhus, a severe mite-borne human disease. Ot is unusual amongst Rickettsiaceae in budding out of infected cells in a process resembling that used by enveloped viruses. Here, we show that Ot bacteria that have budded out of host cells are in a distinct developmental stage compared with intracellular bacteria. We refer to these two stages as intracellular and extracellular bacteria (IB and EB, respectively). These two forms differ in physical properties: IB is elongated, and EB is round. Additionally, IB has higher levels of peptidoglycan and is physically robust compared with EB. The two bacterial forms differentially express proteins involved in bacterial physiology and host-pathogen interactions, specifically those involved in bacterial dormancy and stress response, secreted bacterial effectors, and outer membrane autotransporter proteins ScaA and ScaC. Whilst both populations are infectious, entry of IB Ot is sensitive to inhibitors of both clathrin-mediated endocytosis and micropinocytosis, whereas entry of EB Ot is only sensitive to a micropinocytosis inhibitor. Our identification and detailed characterization of two developmental forms of Ot significantly advances our understanding of the intracellular lifecycle of an important human pathogen.

2022-05-11 | PXD028218 | JPOST Repository

A Bacterial Protein Targets the BAHD1 Chromatin Complex to Stimulate Type III Interferon Response

Project description:Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate IFN-stimulated genes (ISGs). IFN-M-NM-; expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1+/- mice or when lntA was constitutively expressed. Thus the LntA-BAHD1 interplay may modulate IFN-M-NM-;M-bM-^HM-^Rmediated immune response to control bacterial colonization of the host. In this dataset, we include the expression data obtained following infection of human Lovo cells infected with Listeria bacteria expressing (LntA-V5+) or not (DlntA) the virulence facteur LntA RNA from LoVo cells grown in 6-well plates and infected with either lntAV5+ or lntAM-bM-^@M-^S strains was extracted. Three biological replicates were analyzed for each experimental condition.

2011-02-03 | E-GEOD-26414 | biostudies-arrayexpress

A Bacterial Protein Targets the BAHD1 Chromatin Complex to Stimulate Type III Interferon Response

Project description:Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate IFN-stimulated genes (ISGs). IFN-λ expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1+/- mice or when lntA was constitutively expressed. Thus the LntA-BAHD1 interplay may modulate IFN-λ−mediated immune response to control bacterial colonization of the host. In this dataset, we include the expression data obtained following infection of human Lovo cells infected with Listeria bacteria expressing (LntA-V5+) or not (DlntA) the virulence facteur LntA

2011-02-03 | GSE26414 | GEO

Expression data of A2EN cells during early stage of Chlamydia trachomatis infection

Project description:Chlamydia trachomatis is an obligate intracellular pathogen that causes trachoma and sextually transmitted disease in human. During early stage of infection, Chlamydia secreted bacterial effector proteins into host cell cytoplasm to help its entry and estabilishment of early replicated niche. We identified a Chlamydia mutant that lack an early Effector. To address the function of this effector, we infected A2EN cells with this mutant (G1V) and its complemented counterpart (G1TEPP) to see what host gene transcriptions are affected by this effector. A2EN cells were mock infected, or infected with a Chlamydia mutant or its complemented counterpart for 4 hour post infection.

2014-04-09 | E-GEOD-54336 | biostudies-arrayexpress

Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

Project description:Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound “bacteria containing vacuole” (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using Significance Analysis of INTeractome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc, CT226, using bacterial two-hybrid and co-immunoprecipitation assays. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.

2019-10-25 | PXD015883 | Pride

Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

2019-10-25 | PXD015890 | Pride

Expression data of A2EN cells during early stage of Chlamydia trachomatis infection

2014-04-09 | GSE54336 | GEO

Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila

Project description:This SuperSeries is composed of the following subset Series: GSE26473: Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila [exp1] GSE26490: Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila [exp2] Refer to individual Series

2011-01-08 | E-GEOD-26491 | biostudies-arrayexpress

The E. coli type III secreted effectors NleB and EspL interact with the microtubule associated protein ensconsin to disrupt vesicle trafficking, part 1

Project description:Enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC) translocate a set of type III effector proteins into host cells that are critical for bacterial virulence. These effectors subvert normal host pathways by interacting with a variety of targets within the cell, but the binding partners and mechanism of action of the majority of effectors are not understood. We identified the microtubule associated protein, ensconsin, as a novel target for two EPEC/EHEC effectors. We found that the secreted effectors NleB and EspL bind host ensconsin and work synergistically to paralyze kinesin-based intracellular vesicular transport. Our findings demonstrate that EPEC/EHEC encode multiple effectors that control intracellular vesicle movement and suggest a simple strategy for broad-based immobilization of host cells by pathogens.

2016-12-22 | PXD002933 | Pride

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