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The bacterial effector GarD shields Chlamydia trachomatis inclusions from RNF213-mediated ubiquitylation and destruction.

ABSTRACT: Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and a major threat to women's reproductive health in particular. This obligate intracellular pathogen resides and replicates within a cellular compartment termed an inclusion, where it is sheltered by unknown mechanisms from gamma-interferon (IFNγ)-induced cell-autonomous host immunity. Through a genetic screen, we uncovered the Chlamydia inclusion membrane protein gamma resistance determinant (GarD) as a bacterial factor protecting inclusions from cell-autonomous immunity. In IFNγ-primed human cells, inclusions formed by garD loss-of-function mutants become decorated with linear ubiquitin and are eliminated. Leveraging cellular genome-wide association data, we identified the ubiquitin E3 ligase RNF213 as a candidate anti-Chlamydia protein. We demonstrate that IFNγ-inducible RNF213 facilitates the ubiquitylation and destruction of GarD-deficient inclusions. Furthermore, we show that GarD operates as a cis-acting stealth factor barring RNF213 from targeting inclusions, thus functionally defining GarD as an RNF213 antagonist essential for chlamydial growth during IFNγ-stimulated immunity.

SUBMITTER: Walsh SC

PROVIDER: S-EPMC9772000 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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The bacterial effector GarD shields Chlamydia trachomatis inclusions from RNF213-mediated ubiquitylation and destruction.

Walsh Stephen C SC Reitano Jeffrey R JR Dickinson Mary S MS Kutsch Miriam M Hernandez Dulcemaria D Barnes Alyson B AB Schott Benjamin H BH Wang Liuyang L Ko Dennis C DC Kim So Young SY Valdivia Raphael H RH Bastidas Robert J RJ Coers Jörn J

Cell host & microbe 20220908 12

Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and a major threat to women's reproductive health in particular. This obligate intracellular pathogen resides and replicates within a cellular compartment termed an inclusion, where it is sheltered by unknown mechanisms from gamma-interferon (IFNγ)-induced cell-autonomous host immunity. Through a genetic screen, we uncovered the Chlamydia inclusion membrane protein gamma resistance determinant (GarD) as a bac ...[more]

PMID: 36084633

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Project description:The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.

Project description:Mitochondria are critical cellular organelles that perform a wide variety of functions, including energy production and immune regulation. To perform these functions, mitochondria contain approximately 1,500 proteins, the majority of which are encoded in the nuclear genome, translated in the cytoplasm, and translocated to the mitochondria using distinct mitochondrial targeting sequences (MTS). Bacterial proteins can also contain MTS and localize to the mitochondria. For the obligate intracellular human pathogen Chlamydia trachomatis, interaction with various host cell organelles promotes intracellular replication. However, the extent and mechanisms through which Chlamydia cells interact directly with mitochondria remain unclear. We investigated the presence of MTS in the C. trachomatis genome and discovered 30 genes encoding proteins with around 70% or greater probability of mitochondrial localization. Five are translocated to the mitochondria upon ectopic expression in HeLa cells. Mass spectrometry of isolated mitochondria from infected cells revealed that two of these proteins localize to the mitochondria during infection. Comparison of mitochondria from infected and uninfected cells suggests that chlamydial infection affects the mitochondrial protein composition. Around 125 host proteins were significantly decreased or absent in mitochondria from infected cells. Among these were proapoptotic factors and those related to mitochondrial fission/fusion dynamics. Conversely, 82 host proteins were increased in or specific to mitochondria of infected cells, many of which act as antiapoptotic factors and upregulators of cellular metabolism. These data support the notion that C. trachomatis specifically targets host mitochondria to manipulate cell fate decisions and metabolic function to support pathogen survival and replication. IMPORTANCE Obligate intracellular bacteria have evolved multiple means to promote their intracellular survival and replication within the otherwise harsh environment of the eukaryotic cell. Nutrient acquisition and avoidance of cellular defense mechanisms are critical to an intracellular lifestyle. Mitochondria are critical organelles that produce energy in the form of ATP and regulate programmed cell death responses to invasive pathogenic microbes. Cell death prior to completion of replication would be detrimental to the pathogen. C. trachomatis produces at least two and possibly more proteins that target the mitochondria. Collectively, C. trachomatis infection modulates the mitochondrial protein composition, favoring a profile suggestive of downregulation of apoptosis.

Dataset Information

The bacterial effector GarD shields Chlamydia trachomatis inclusions from RNF213-mediated ubiquitylation and destruction.

Publications

The bacterial effector GarD shields Chlamydia trachomatis inclusions from RNF213-mediated ubiquitylation and destruction.

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