Leigh A. Baxt

Bacterial subversion of host innate immune pathways.

Defense and Counter-Defense Provided a pathogen can enter the body and survive coughing and spluttering, peristalsis, and mucus, the first active responses the host evokes to an invading organism will be at the level of the first cell encountered, well before classical cellular immunity and antibody responses are initiated. Randow et al. (p. 701) review the range of intracellular defenses against incoming pathogens and describe how compartmental boundaries within the cell provide multiple levels at which pathogens can be thwarted in their attempts to subjugate the cell to do their bidding. Baxt et al. (p. 697) review the range of evasion tactics that bacterial pathogens can summon to counter host repulsion and establish a niche in which to replicate and ensure onward transmission. The pathogenesis of infection is a continuously evolving battle between the human host and the infecting microbe. The past decade has brought a burst of insights into the molecular mechanisms of innate immune responses to bacterial pathogens. In parallel, multiple specific mechanisms by which microorganisms subvert these host responses have been uncovered. This Review highlights recently characterized mechanisms by which bacterial pathogens avoid killing by innate host responses, including autophagy pathways and a proinflammatory cytokine transcriptional response, and by the manipulation of vesicular trafficking to avoid the toxicity of lysosomal enzymes.

Host and Bacterial Proteins That Repress Recruitment of LC3 to Shigella Early during Infection

Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min.), the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4–6 hr) by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG). Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.

Role of Autophagy in the Maintenance of Intestinal Homeostasis

Genome-wide association studies of inflammatory bowel disease have identified several risk loci in genes that regulate autophagy, and studies have provided insight into the functional effects of these polymorphisms. We review the mechanisms by which autophagy contributes to intestinal homeostasis, focusing on its cell type-specific roles in regulating gut ecology, restricting pathogenic bacteria, and controlling inflammation. Based on this information, we are beginning to understand how alterations in autophagy can contribute to intestinal inflammation.

Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense.

Integrated genomics of crohn's disease risk variant identifies a role for CLEC12A in antibacterial autophagy

Summary The polymorphism ATG16L1 T300A, associated with increased risk of Crohn’s disease, impairs pathogen defense mechanisms including selective autophagy, but specific pathway interactions altered by the risk allele remain unknown. Here, we use perturbational profiling of human peripheral blood cells to reveal that CLEC12A is regulated in an ATG16L1-T300A-dependent manner. Antibacterial autophagy is impaired in CLEC12A-deficient cells, and this effect is exacerbated in the presence of the ATG16L1∗300A risk allele. Clec12a−/− mice are more susceptible to Salmonella infection, supporting a role for CLEC12A in antibacterial defense pathways in vivo. CLEC12A is recruited to sites of bacterial entry, bacteria-autophagosome complexes, and sites of sterile membrane damage. Integrated genomics identified a functional interaction between CLEC12A and an E3-ubiquitin ligase complex that functions in antibacterial autophagy. These data identify CLEC12A as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease.

Intermediate filaments enable pathogen docking to trigger type 3 effector translocation

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen S. flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.

Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins

Background: PDZ domains provide specificity in protein-protein interactions in eukaryotic systems. Results: Shigella OspE, which is delivered into human cells during infection, binds PDLIM7 via a PDZ interaction and contributes to activation of PKC. Conclusion: During infection, OspE contributes to activation of PKC through PDZ-mediated binding to PDLIM7. Significance: PDZ interactions likely contribute to pathogenesis of several bacterial pathogens. Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.

Anaerobic environment of the intestine primes pathogenic Shigella for infection.

Evaluation of: Marteyn B, West NP, Browning DF et al. Modulation of Shigella virulence in response to available oxygen in vivo. Nature 465, 355–358 (2010). Marteyn et al. have investigated the role of oxygen and the regulator FNR in infection by the intracellular enteric pathogen Shigella flexneri. FNR is active under anaerobic conditions like those present in the lumen of the distal intestine. FNR causes elongation of a secretion apparatus required for bacterial entry into cells and represses secretion of proteins that trigger entry. Higher oxygen levels present at the intestinal cell surface are sufficient to inactivate FNR, thereby derepressing secretion. Thus, bacteria are ‘primed’ in the anaerobic environment of the lumen, and entry is triggered by the aerobic conditions at the intestinal cell surface. FNR is conserved among many enteric pathogens, suggesting that regulation of virulence in response to oxygen may be widely conserved.

Systematic analysis of bacterial effector-PDZ interactions demonstrates Shigella OspE promotes PKC activation via PDLIM proteins

Background: PDZ domains provide specificity in protein-protein interactions in eukaryotic systems. Results: Shigella OspE, which is delivered into human cells during infection, binds PDLIM7 via a PDZ interaction and contributes to activation of PKC. Conclusion: During infection, OspE contributes to activation of PKC through PDZ-mediated binding to PDLIM7. Significance: PDZ interactions likely contribute to pathogenesis of several bacterial pathogens. Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.

Systematic analysis of bacterial effector-postsynaptic density 95/discs large/zonula occludens-1 (PDZ) interactions demonstrates Shigella OspE promotes PKC activation via PDLIM proteins

Background: PDZ domains provide specificity in protein-protein interactions in eukaryotic systems. Results: Shigella OspE, which is delivered into human cells during infection, binds PDLIM7 via a PDZ interaction and contributes to activation of PKC. Conclusion: During infection, OspE contributes to activation of PKC through PDZ-mediated binding to PDLIM7. Significance: PDZ interactions likely contribute to pathogenesis of several bacterial pathogens. Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.