A FACS-Based Genome-wide CRISPR Screen Reveals a Requirement for COPI in Chlamydia trachomatis Invasion

Park, Joseph S.; Helble, Jennifer D.; Lazarus, Jacob E.; Yang, Guanhua; Blondel, Carlos J.; Doench, John G.; Starnbach, Michael N.; Waldor, Matthew K.

doi:10.1016/j.isci.2018.12.011

Cited by 23 publications

(18 citation statements)

References 82 publications

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“…While N-WASP and the ARP2/3 complex clearly play an integral role in chlamydial invasion, various screens have identified other factors including cortactin, Abl kinase, WAVE2, SNX9, and COPI [ 8 , 26 , 43 – 45 ] that play a role in chlamydial invasion. SNX9 mediates F-actin rearrangements and filopodia formation through associations with N-WASP [ 46 ].…”

Section: Discussionmentioning

confidence: 99%

The Chlamydia trachomatis secreted effector TmeA hijacks the N-WASP-ARP2/3 actin remodeling axis to facilitate cellular invasion

et al. 2020

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As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain illdefined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.

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Section: Discussionmentioning

confidence: 99%

The Chlamydia trachomatis secreted effector TmeA hijacks the N-WASP-ARP2/3 actin remodeling axis to facilitate cellular invasion

et al. 2020

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“…4 ). This is reminiscent of the situation in epithelial cells, in which COPI promotes C. trachomatis entry 39 and COPI vesicles 40 might deliver nutrients to chlamydial inclusions 41 . ADP-ribosylation factor 1 (Arf1), a key regulator of membrane organization, localizes to chlamydial inclusions ( C. trachomatis , C. muridarum and C. pneumoniae ) 59 and regulates the recruitment of COPI vesicles 60 .…”

Section: Discussionmentioning

confidence: 89%

“…The coat protein complex I (COPI) seems to promote C. trachomatis entry into the host cell downstream of cell surface attachment 39 . A critical role of COPI in chlamydial infection is also shown by the identification of α, β, β′, γ and ξ-COP as important host factors in C. caviae infection 40 .…”

Section: Resultsmentioning

confidence: 99%

NK Cell-Mediated Processing Of Chlamydia psittaci Drives Potent Anti-Bacterial Th1 Immunity

Radomski

Franzke

Matthiesen

et al. 2019

Sci Rep

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Natural killer (NK) cells are innate immune cells critically involved in the early immune response against various pathogens including chlamydia. Here, we demonstrate that chlamydia-infected NK cells prevent the intracellular establishment and growth of the bacteria. Upon infection, they display functional maturation characterized by enhanced IFN-γ secretion, CD146 induction, PKCϴ activation, and granule secretion. Eventually, chlamydia are released in a non-infectious, highly immunogenic form driving a potent Th1 immune response. Further, anti-chlamydial antibodies generated during immunization neutralize the infection of epithelial cells. The release of chlamydia from NK cells requires pKCϴ function and active degranulation, while granule-associated granzyme B drives the loss of chlamydial infectivity. Cellular infection and bacterial release can be undergone repeatedly and do not affect NK cell function. Strikingly, NK cells passing through such an infection cycle significantly improve their cytotoxicity. Thus, NK cells not only protect themselves against productive chlamydial infections but also actively trigger potent anti-bacterial responses. NK cells play an important role in the immune response against various pathogens including chlamydia 1. Through their interactions with other immune cells, they are important mediators between innate and adaptive immunity 2. NK cells express a set of activating/inhibiting receptors 3 , which generate signals whose balance determines which cellular program is chosen 4. They are activated by various cytokines 5 resulting in the activation of phospholipase C (PLC). PLC generates two messengers, 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which activate protein kinases C (PKCs) and mobilize Ca 2+ from intracellular stores. DAG promotes PKCϴ translocation to membranes and phospho-activation, regulating NK-mediated effector functions 6. To detect and lyse target cells, NK cells use distinct mechanisms: Antibody-dependent cell-mediated cytotoxicity (ADCC) and natural cytotoxic activity 7. In ADCC, the Fc part of target cell-bound IgG is recognized by the FcγRΙΙΙ receptor (CD16) on NK cells, upon which cytotoxic proteins are released in addition to IFN-γ. This leads to the cytotoxic killing of target cells 8. No prior sensitization is needed for natural cytotoxicity, allowing for rapid detection/killing by this mechanism 8. After direct contact with the target cell, secretory granules (containing granzymes and perforin) are released into the immunological gap 8. Moreover, NK cells can kill via TNF family ligands 9 as well as via the secretion of cytokines and chemokines 10. DAG-mediated activation of PKCs is sufficient to induce degranulation of NK cells, leading to the release of granzyme B 11. Granzyme B is initially synthesized as an inactive precursor whose propeptide is removed by cathepsin C 12 , generating the enzymatically active protease. Perforin mediates the entry of activated granzyme B into the cytoplasm of target cells, where a large number o...

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“…For example, in an infected cell, post-Golgi vesicles are rerouted to deliver host sphingolipids and cholesterol, but not proteins, to the inclusion and the bacteria themselves (18)(19)(20). Vesicles that mediate anterograde and retrograde transport between the ER and the Golgi are also important for the infection (21)(22)(23).…”

Section: Downloaded Frommentioning

confidence: 99%

The Small Molecule H89 Inhibits Chlamydia Inclusion Growth and Production of Infectious Progeny

et al. 2021

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Chlamydia is an obligate intracellular bacterium and the most common reportable cause of human infection in the U.S. This pathogen proliferates inside a eukaryotic host cell, where it resides within a membrane-bound compartment called the chlamydial inclusion. It has an unusual developmental cycle, marked by conversion between a replicating form, the reticulate body (RB), and an infectious form, the elementary body (EB). We found that the small molecule H89 slowed inclusion growth and decreased overall RB replication by 2-fold, but caused a 25-fold reduction in infectious EBs. This disproportionate effect on EB production was mainly due to a defect in RB-to-EB conversion and not to the induction of chlamydial persistence, which is an altered growth state. Although H89 is a known inhibitor of specific protein kinases and vesicular transport to and from the Golgi, it did not cause these anti-chlamydial effects by blocking the protein kinases PKA or PKC, or by inhibiting protein or lipid transport. H89 is thus a novel anti-chlamydial compound that has a unique combination of effects on the intracellular Chlamydia infection.

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A FACS-Based Genome-wide CRISPR Screen Reveals a Requirement for COPI in Chlamydia trachomatis Invasion

Cited by 23 publications

References 82 publications

The Chlamydia trachomatis secreted effector TmeA hijacks the N-WASP-ARP2/3 actin remodeling axis to facilitate cellular invasion

The Chlamydia trachomatis secreted effector TmeA hijacks the N-WASP-ARP2/3 actin remodeling axis to facilitate cellular invasion

NK Cell-Mediated Processing Of Chlamydia psittaci Drives Potent Anti-Bacterial Th1 Immunity

The Small Molecule H89 Inhibits Chlamydia Inclusion Growth and Production of Infectious Progeny

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