Ku70 senses cytosolic DNA and assembles a tumor-suppressive signalosome.

The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5+Rab7+ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.

Clostridium perfringens virulence factors are nonredundant activators of the NLRP3 inflammasome.

Inflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogen Clostridium perfringens are nonredundant activators of the NLRP3 inflammasome in mice and humans. C. perfringens lecithinase (also known as phospolipase C) and C. perfringens perfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1+ vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome-dependent cytokines IL-1ß and IL-18, and the induction of cell death independently of the pore-forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin-1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasome in vivo and that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase-induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation during C. perfringens infection and that this mode of action can be similarly exploited for sensing by a single inflammasome.

Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes.

Moraxella catarrhalis is an important human respiratory pathogen and a major causative agent of otitis media and chronic obstructive pulmonary disease. Toll-like receptors contribute to, but cannot fully account for, the complexity of the immune response seen in M. catarrhalis infection. Using primary mouse bone marrow-derived macrophages to examine the host response to M. catarrhalis infection, our global transcriptomic and targeted cytokine analyses revealed activation of immune signalling pathways by both membrane-bound and cytosolic pattern-recognition receptors. We show that M. catarrhalis and its outer membrane vesicles or lipooligosaccharide (LOS) can activate the cytosolic innate immune sensor caspase-4/11, gasdermin-D-dependent pyroptosis, and the NLRP3 inflammasome in human and mouse macrophages. This pathway is initiated by type I interferon signalling and guanylate-binding proteins (GBPs). We also show that inflammasomes and GBPs, particularly GBP2, are required for the host defence against M. catarrhalis in mice. Overall, our results reveal an essential role for the interferon-inflammasome axis in cytosolic recognition and immunity against M. catarrhalis, providing new molecular targets that may be used to mitigate pathological inflammation triggered by this pathogen.

Differential activation of NLRP3 inflammasome by Acinetobacter baumannii strains.

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A. baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1ß and IL-18, the recruitment of innate immune effectors against A. baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the host's innate response and activation of the inflammasome during A. baumannii infection. In this study, we compared nine A. baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death in primary macrophages, epithelial lung cell line and mice. We found a variation in survival outcomes of mice and bacterial dissemination in organs among three commercially available A. baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found variability among A. baumannii strains in activation of the NLRP3 inflammasome, non-canonical Caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokine IL-1ß and programmed cell death. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with different virulence to investigate the innate immune response to A. baumannii infection.

Pathogen-selective killing by guanylate-binding proteins as a molecular mechanism leading to inflammasome signaling.

Inflammasomes are cytosolic signaling complexes capable of sensing microbial ligands to trigger inflammation and cell death responses. Here, we show that guanylate-binding proteins (GBPs) mediate pathogen-selective inflammasome activation. We show that mouse GBP1 and GBP3 are specifically required for inflammasome activation during infection with the cytosolic bacterium Francisella novicida. We show that the selectivity of mouse GBP1 and GBP3 derives from a region within the N-terminal domain containing charged and hydrophobic amino acids, which binds to and facilitates direct killing of F. novicida and Neisseria meningitidis, but not other bacteria or mammalian cells. This pathogen-selective recognition by this region of mouse GBP1 and GBP3 leads to pathogen membrane rupture and release of intracellular content for inflammasome sensing. Our results imply that GBPs discriminate between pathogens, confer activation of innate immunity, and provide a host-inspired roadmap for the design of synthetic antimicrobial peptides that may be of use against emerging and re-emerging pathogens.

Clostridium septicum α-toxin activates the NLRP3 inflammasome by engaging GPI-anchored proteins.

Clostridium species are a group of Gram-positive bacteria that cause diseases in humans, such as food poisoning, botulism, and tetanus. Here, we analyzed 10 different Clostridium species and identified that Clostridium septicum, a pathogen that causes sepsis and gas gangrene, activates the mammalian cytosolic inflammasome complex in mice and humans. Mechanistically, we demonstrate that α-toxin secreted by C. septicum binds to glycosylphosphatidylinositol (GPI)-anchored proteins on the host plasma membrane, oligomerizing and forming a membrane pore that is permissive to efflux of magnesium and potassium ions. Efflux of these cytosolic ions triggers the activation of the innate immune sensor NLRP3, inducing activation of caspase-1 and gasdermin D, secretion of the proinflammatory cytokines interleukin-1ß and interleukin-18, pyroptosis, and plasma membrane rupture via ninjurin-1. Furthermore, α-toxin of C. septicum induces rapid inflammasome-mediated lethality in mice and pharmacological inhibition of the NLRP3 inflammasome using MCC950 prevents C. septicum-induced lethality. Overall, our results reveal that cytosolic innate sensing of α-toxin is central to the recognition of C. septicum infection and that therapeutic blockade of the inflammasome pathway may prevent sepsis and death caused by toxin-producing pathogens.

Bacillus cereus: Epidemiology, Virulence Factors, and Host-Pathogen Interactions.

The toxin-producing bacterium Bacillus cereus is an important and neglected human pathogen and a common cause of food poisoning. Several toxins have been implicated in disease, including the pore-forming toxins hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE). Recent work revealed that HBL binds to the mammalian surface receptors LITAF and CDIP1 and that both HBL and NHE induce potassium efflux and activate the NLRP3 inflammasome, leading to pyroptosis. These mammalian receptors, in part, contribute to inflammation and pathology. Other putative virulence factors of B. cereus include cytotoxin K, cereulide, metalloproteases, sphingomyelinase, and phospholipases. In this review, we highlight the latest progress in our understanding of B. cereus biology, epidemiology, and pathogenesis, and discuss potential new directions for research in this field.

Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome.

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome.

A multicomponent toxin from Bacillus cereus incites inflammation and shapes host outcome via the NLRP3 inflammasome.

Host recognition of microbial components is essential in mediating an effective immune response. Cytosolic bacteria must secure entry into the host cytoplasm to facilitate replication and, in doing so, liberate microbial ligands that activate cytosolic innate immune sensors and the inflammasome. Here, we identified a multicomponent enterotoxin, haemolysin BL (HBL), that engages activation of the inflammasome. This toxin is highly conserved among the human pathogen Bacillus cereus. The three subunits of HBL bind to the cell membrane in a linear order, forming a lytic pore and inducing activation of the NLRP3 inflammasome, secretion of interleukin-1ß and interleukin-18, and pyroptosis. Mechanistically, the HBL-induced pore results in the efflux of potassium and triggers the activation of the NLRP3 inflammasome. Furthermore, HBL-producing B. cereus induces rapid inflammasome-mediated mortality. Pharmacological inhibition of the NLRP3 inflammasome using MCC950 prevents B. cereus-induced lethality. Overall, our results reveal that cytosolic sensing of a toxin is central to the innate immune recognition of infection. Therapeutic modulation of this pathway enhances host protection against deadly bacterial infections.

Cytosolic Recognition of Microbes and Pathogens: Inflammasomes in Action.

Infection is a dynamic biological process underpinned by a complex interplay between the pathogen and the host. Microbes from all domains of life, including bacteria, viruses, fungi, and protozoan parasites, have the capacity to cause infection. Infection is sensed by the host, which often leads to activation of the inflammasome, a cytosolic macromolecular signaling platform that mediates the release of the proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 and cleavage of the pore-forming protein gasdermin D, leading to pyroptosis. Host-mediated sensing of the infection occurs when pathogens inject or carry pathogen-associated molecular patterns (PAMPs) into the cytoplasm or induce damage that causes cytosolic liberation of danger-associated molecular patterns (DAMPs) in the host cell. Recognition of PAMPs and DAMPs by inflammasome sensors, including NLRP1, NLRP3, NLRC4, NAIP, AIM2, and Pyrin, initiates a cascade of events that culminate in inflammation and cell death. However, pathogens can deploy virulence factors capable of minimizing or evading host detection. This review presents a comprehensive overview of the mechanisms of microbe-induced activation of the inflammasome and the functional consequences of inflammasome activation in infectious diseases. We also explore the microbial strategies used in the evasion of inflammasome sensing at the host-microbe interaction interface.

Molecular mechanisms of inflammasome signaling.

The inflammasome is a macromolecular protein complex that mediates proteolytic cleavage of pro-IL-1ß and -IL-18 and induces cell death in the form of pyroptosis. Certain nucleotide-binding oligomerization domain-like receptors (NLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), or tripartite motif (TRIM) family receptors trigger the assembly of an inflammasome in response to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Recent studies have revealed a multitude of host components and signals that are essential for controlling canonical and noncanonical inflammasome activation and pyroptosis. These include pore-forming gasdermin proteins, the never in mitosis A-related kinase 7 (NEK7), IFN-inducible proteins (IFIs), reactive oxygen species (ROS), autophagy, potassium efflux, mitochondrial perturbations, and microbial metabolites. Here, we provide a comprehensive overview of the molecular and signaling mechanisms that provide stringent regulation over the activation and effector functions of the inflammasome.