LPS-aggregating proteins GBP1 and GBP2 are each sufficient to enhance caspase-4 activation both in cellulo and in vitro.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates "free" LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an in vitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock.

Inflammation associated with gram-negative bacterial infections is often instigated by the bacterial cell wall component lipopolysaccharide (LPS). LPS-induced inflammation and resulting life-threatening sepsis are mediated by the two distinct LPS receptors TLR4 and caspase-11 (caspase-4/-5 in humans). Whereas the regulation of TLR4 activation by extracellular and phago-endosomal LPS has been studied in great detail, auxiliary host factors that specifically modulate recognition of cytosolic LPS by caspase-11 are largely unknown. This study identifies autophagy-related and dynamin-related membrane remodeling proteins belonging to the family of Immunity-related GTPases M clade (IRGM) as negative regulators of caspase-11 activation in macrophages. Phagocytes lacking expression of mouse isoform Irgm2 aberrantly activate caspase-11-dependent inflammatory responses when exposed to extracellular LPS, bacterial outer membrane vesicles, or gram-negative bacteria. Consequently, Irgm2-deficient mice display increased susceptibility to caspase-11-mediated septic shock in vivo. This Irgm2 phenotype is partly reversed by the simultaneous genetic deletion of the two additional Irgm paralogs Irgm1 and Irgm3, indicating that dysregulated Irgm isoform expression disrupts intracellular LPS processing pathways that limit LPS availability for caspase-11 activation.

A Rapidly Evolving Polybasic Motif Modulates Bacterial Detection by Guanylate Binding Proteins.

Cell-autonomous immunity relies on the rapid detection of invasive pathogens by host proteins. Guanylate binding proteins (GBPs) have emerged as key mediators of vertebrate immune defense through their ability to recognize a diverse array of intracellular pathogens and pathogen-containing cellular compartments. Human and mouse GBPs have been shown to target distinct groups of microbes, although the molecular determinants of pathogen specificity remain unclear. We show that rapid diversification of a C-terminal polybasic motif (PBM) in primate GBPs controls recognition of the model cytosolic bacterial pathogen Shigella flexneri By swapping this membrane-binding motif between primate GBP orthologs, we found that the ability to target S. flexneri has been enhanced and lost in specific lineages of New World primates. Single substitutions in rapidly evolving sites of the GBP1 PBM are sufficient to abolish or restore bacterial detection abilities, illustrating a role for epistasis in the evolution of pathogen recognition. We further demonstrate that the squirrel monkey GBP2 C-terminal domain recently gained the ability to target S. flexneri through a stepwise process of convergent evolution. These findings reveal a mechanism by which accelerated evolution of a PBM shifts GBP target specificity and aid in resolving the molecular basis of GBP function in cell-autonomous immune defense.IMPORTANCE Many infectious diseases are caused by microbes that enter and survive within host cells. Guanylate binding proteins (GBPs) are a group of immune proteins which recognize and inhibit a variety of intracellular pathogenic microbes. We discovered that a short sequence within GBPs required for the detection of bacteria, the polybasic motif (PBM), has been rapidly evolving between primate species. By swapping PBMs between primate GBP1 genes, we were able to show that specific sequences can both reduce and improve the ability of GBP1 to target intracellular bacteria. We also show that the ability to envelop bacteria has independently evolved in GBP2 of South American monkeys. Taking the results together, this report illustrates how primate GBPs have adapted to defend against infectious pathogens.

Detection of Cytosolic Shigella flexneri via a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility.

Dynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN-γ)-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri Rough lipopolysaccharide (LPS) mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6). GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis These studies reveal that human GBP1 uniquely functions as an intracellular "glue trap," inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment.IMPORTANCE Several pathogenic bacterial species evolved to invade, reside in, and replicate inside the cytosol of their host cells. One adaptation common to most cytosolic bacterial pathogens is the ability to coopt the host's actin polymerization machinery in order to generate force for intracellular movement. This actin-based motility enables Gram-negative bacteria, such as Shigella species, to propel themselves into neighboring cells, thereby spreading from host cell to host cell without exiting the intracellular environment. Here, we show that the human protein GBP1 acts as a cytosolic "glue trap," capturing cytosolic Gram-negative bacteria through a unique protein motif and preventing disseminated infections in cell culture models. To escape from this GBP1-mediated host defense, Shigella employs a virulence factor that prevents or dislodges the association of GBP1 with cytosolic bacteria. Thus, therapeutic strategies to restore GBP1 binding to Shigella may lead to novel treatment options for shigellosis in the future.

Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems.

Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as "patterns of pathogenesis" associated with PVs. We report that the delivery of GBP2 to Legionella-containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia-containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella-containing vacuoles or YCVs is substantially diminished in Galectin-3-deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3-dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.

Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells.

The cytokine gamma interferon (IFN-γ) induces cell-autonomous immunity to combat infections with intracellular pathogens, such as the bacterium Chlamydia trachomatis The present study demonstrates that IFN-γ-primed human cells ubiquitinate and eliminate intracellular Chlamydia-containing vacuoles, so-called inclusions. We previously described how IFN-γ-inducible immunity-related GTPases (IRGs) employ ubiquitin systems to mark inclusions for destruction in mouse cells and, furthermore, showed that the rodent pathogen Chlamydia muridarum blocks ubiquitination of its inclusions by interfering with mouse IRG function. Here, we report that ubiquitination of inclusions in human cells is independent of IRG and thus distinct from the murine pathway. We show that C. muridarum is susceptible to inclusion ubiquitination in human cells, while the closely related human pathogen C. trachomatis is resistant. C. muridarum, but not C. trachomatis, inclusions attract several markers of cell-autonomous immunity, including the ubiquitin-binding protein p62, the ubiquitin-like protein LC3, and guanylate-binding protein 1. Consequently, we find that IFN-γ priming of human epithelial cells triggers the elimination of C. muridarum, but not C. trachomatis, inclusions. This newly described defense pathway is independent of indole-2,3-dioxygenase, a known IFN-γ-inducible anti-Chlamydia resistance factor. Collectively, our observations indicate that C. trachomatis evolved mechanisms to avoid a human-specific, ubiquitin-mediated response as part of its unique adaptation to its human host. IMPORTANCE: Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and responsible for significant morbidity, including pelvic inflammatory disease, infertility, and ectopic pregnancies in women. As an obligate intracellular pathogen, C. trachomatis is in perpetual conflict with cell-intrinsic defense programs executed by its human host. Our study defines a novel anti-Chlamydia host resistance pathway active in human epithelial cells. This defense program promotes the deposition of the small antimicrobial protein ubiquitin on vacuoles containing Chlamydia We show that this ubiquitin-based resistance pathway of human cells is highly effective against a Chlamydia species adapted to rodents but ineffective against human-adapted C. trachomatis This observation indicates that C. trachomatis evolved strategies to avoid entrapment within ubiquitin-labeled vacuoles as part of its adaptation to the human innate immune system.

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins.

Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association of Toxoplasma- and Chlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.

The E2-like conjugation enzyme Atg3 promotes binding of IRG and Gbp proteins to Chlamydia- and Toxoplasma-containing vacuoles and host resistance.

Cell-autonomous immunity to the bacterial pathogen Chlamydia trachomatis and the protozoan pathogen Toxoplasma gondii is controlled by two families of Interferon (IFN)-inducible GTPases: Immunity Related GTPases (IRGs) and Guanylate binding proteins (Gbps). Members of these two GTPase families associate with pathogen-containing vacuoles (PVs) and solicit antimicrobial resistance pathways specifically to the intracellular site of infection. The proper delivery of IRG and Gbp proteins to PVs requires the autophagy factor Atg5. Atg5 is part of a protein complex that facilitates the transfer of the ubiquitin-like protein Atg8 from the E2-like conjugation enzyme Atg3 to the lipid phosphatidylethanolamine. Here, we show that Atg3 expression, similar to Atg5 expression, is required for IRG and Gbp proteins to dock to PVs. We further demonstrate that expression of a dominant-active, GTP-locked IRG protein variant rescues the PV targeting defect of Atg3- and Atg5-deficient cells, suggesting a possible role for Atg proteins in the activation of IRG proteins. Lastly, we show that IFN-induced cell-autonomous resistance to C. trachomatis infections in mouse cells depends not only on Atg5 and IRG proteins, as previously demonstrated, but also requires the expression of Atg3 and Gbp proteins. These findings provide a foundation for a better understanding of IRG- and Gbp-dependent cell-autonomous resistance and its regulation by Atg proteins.

IRG and GBP host resistance factors target aberrant, "non-self" vacuoles characterized by the missing of "self" IRGM proteins.

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with "non-self" PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on "self" organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of "self" IRGM proteins from these structures.

Small region of Rtf1 protein can substitute for complete Paf1 complex in facilitating global histone H2B ubiquitylation in yeast.

Histone modifications regulate transcription by RNA polymerase II and maintain a balance between active and repressed chromatin states. The conserved Paf1 complex (Paf1C) promotes specific histone modifications during transcription elongation, but the mechanisms by which it facilitates these marks are undefined. We previously identified a 90-amino acid region within the Rtf1 subunit of Paf1C that is necessary for Paf1C-dependent histone modifications in Saccharomyces cerevisiae. Here we show that this histone modification domain (HMD), when expressed as the only source of Rtf1, can promote H3 K4 and K79 methylation and H2B K123 ubiquitylation in yeast. The HMD can restore histone modifications in rtf1Δ cells whether or not it is directed to DNA by a fusion to a DNA binding domain. The HMD can facilitate histone modifications independently of other Paf1C subunits and does not bypass the requirement for Rad6-Bre1. The isolated HMD localizes to chromatin, and this interaction requires residues important for histone modification. When expressed outside the context of full-length Rtf1, the HMD associates with and causes Paf1C-dependent histone modifications to appear at transcriptionally inactive loci, suggesting that its function has become deregulated. Finally, the Rtf1 HMDs from other species can function in yeast. Our findings suggest a direct and conserved role for Paf1C in coupling histone modifications to transcription elongation.