Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling

Toll-like receptor signaling and subsequent activation of NF-κB– and MAPK-dependent genes during infection play an important role in antimicrobial host defense. The YopJ protein of pathogenic Yersinia species inhibits NF-κB and MAPK signaling, resulting in blockade of NF-κB–dependent cytokine production and target cell death. Nevertheless, Yersinia infection induces inflammatory responses in vivo. Moreover, increasing the extent of YopJ-dependent cytotoxicity induced by Yersinia pestis and Yersinia pseudotuberculosis paradoxically leads to decreased virulence in vivo, suggesting that cell death promotes anti-Yersinia host defense. However, the specific pathways responsible for YopJ-induced cell death and how this cell death mediates immune defense against Yersinia remain poorly defined. YopJ activity induces processing of multiple caspases, including caspase-1, independently of inflammasome components or the adaptor protein ASC. Unexpectedly, caspase-1 activation in response to the activity of YopJ required caspase-8, receptor-interacting serine/threonine kinase 1 (RIPK1), and Fas-associated death domain (FADD), but not RIPK3. Furthermore, whereas RIPK3 deficiency did not affect YopJ-induced cell death or caspase-1 activation, deficiency of both RIPK3 and caspase-8 or FADD completely abrogated Yersinia-induced cell death and caspase-1 activation. Mice lacking RIPK3 and caspase-8 in their hematopoietic compartment showed extreme susceptibility to Yersinia and were deficient in monocyte and neutrophil-derived production of proinflammatory cytokines. Our data demonstrate for the first time to our knowledge that RIPK1, FADD, and caspase-8 are required for YopJ-induced cell death and caspase-1 activation and suggest that caspase-8–mediated cell death overrides blockade of immune signaling by YopJ to promote anti-Yersinia immune defense.The innate immune response forms the first line of defense against pathogens. Microbial infection triggers the activation of pattern recognition receptors, such as Toll-like receptors (TLRs) on the cell surface or cytosolic nucleotide binding domain leucine-rich repeat family proteins (NLRs) (1). TLRs induce NF-κB and MAPK signaling to direct immune gene expression, whereas certain NLRs direct the assembly of multiprotein complexes known as inflammasomes that provide platforms for caspase-1 or -11 activation (2). Active caspase-1 and -11 mediate cleavage and secretion of the IL-1 family of proteins and a proinflammatory cell death termed pyroptosis. However, microbial pathogens can interfere with various aspects of innate immune signaling, and the mechanisms that mediate effective immune responses against such pathogens remain poorly understood. Pathogenic Yersiniae cause diseases from gastroenteritis to plague and inject a virulence factor known as YopJ, which inhibits NF-κB and MAPK signaling pathways in target cells (2–4). YopJ activity inhibits proinflammatory cytokine production (4) and induces target cell death (5). YopJ activity induces processing of multiple caspases, including caspases-8, -3, -7, and -1 (6–8). Nevertheless, Yersinia-infected cells exhibit properties of both apoptosis and necrosis (9, 10), and no specific cellular factors have been identified as being absolutely required for YopJ-induced caspase activation and cell death. We previously found that the inflammasome proteins NLR CARD 4 (NLRC4), NLR Pyrin 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC), are dispensable for YopJ-induced caspase-1 processing and cell death (11). Thus, additional pathways likely mediate YopJ-induced caspase-1 activation and cell death.Death receptors, such as TNF receptor and Fas, mediate caspase-8–dependent apoptosis via a death-inducing signaling complex containing receptor-interacting serine/threonine kinase 1 (RIPK1), caspase-8, and Fas-associated death domain (FADD) (12, 13). Whether these proteins are required for Yersinia-induced cell death, and whether this death contributes to antibacterial immune responses, is not known. The Ripoptosome complex, which contains RIPK1, FADD, caspase-8, as well as RIPK3 and cFLIP, regulates apoptosis, programmed necrosis, and survival in response to various stimuli including signaling by the TLR adaptor TRIF (14, 15). Because YopJ-induced cell death is inhibited in the absence of either TLR4 or TRIF (17) we sought to determine whether YopJ-dependent cell death and caspase-1 activation is regulated by caspase-8 or RIPK3 and to define the role of YopJ-dependent cell death in host defense. Here, we describe a previously unappreciated requirement for RIPK1, FADD, and caspase-8, but not RIPK3, in YopJ-induced caspase-1 activation and cell death. Critically, loss of caspase-8 in the hematopoietic compartment resulted in a failure of innate immune cells to produce proinflammatory cytokines in response to Yersinia infection and severely compromised resistance against Yersinia infection. Our data suggest that caspase-8–mediated cell death in response to blockade of NF-κB/MAPKs by YopJ allows for activation of host defense against Yersinia infection. This cell death may thus enable the immune system to override inhibition of immune signaling by microbial pathogens.     

Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results in postnatal lethality, but precisely why Ripk1^−/− mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreER^T2Ripk1^fl/fl mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Vav-iCre Ripk1^fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1^fl/fl mice, showing that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. However, the Vav-iCre Ripk1^fl/fl Ripk3^−/− progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.The proinflammatory cytokine TNF stimulates receptor-interacting serine/threonine-protein kinase 1 (RIPK1) ubiquitination, NFκB and MAPK activation, and induction of apoptosis or necroptosis (1, 2). TNF signaling via TNF receptor 1 (TNFR1) is highly regulated and results in the recruitment of several adapter proteins including TNFR1-associated death domain (TRADD) protein, the E3 ubiquitin ligases cellular inhibitor of apoptosis protein-1 and -2 (cIAP1/2), and TNFR-associated factor 2 (TRAF2) or 5, and the serine threonine death domain-containing kinase RIPK1 (complex I) (1). We have demonstrated that the kinase activity of RIPK1 is not required for NFκB activation (3); rather, RIPK1 is modified by the addition of Lys63-linked and linear polyubiquitin chains (3–6). Polyubiquitinated RIPK1 then recruits NEMO/IκB kinase-γ (IKKγ) to mediate IKK activation and TAK1/TAB2/3 to mediate MAPK activation, resulting in antiapoptotic and proinflammatory gene expression (7, 8). Deubiquitination of RIPK1 by cylindromatosis (CYLD) results in the formation of a cytosolic complex containing TRADD, Fas-associated death domain protein (FADD), caspase-8, and RIPK1 (complex IIa) (2). Caspase-8 cleaves and inactivates RIPK1 and CYLD and stimulates apoptosis (9–11). In the absence of caspase-8 or the presence of caspase inhibitors, TNF family members and potentially other ligands stimulate the kinase activity of RIPK1 to induce necroptosis (9, 11–16). RIPK1 also is recruited to the Toll-like receptor adapter TRIF via the Rip homotypic interaction motif (RHIM) to mediate NFκB activation (17) and, under conditions of caspase-8 inhibition, initiates necroptosis (14, 16). Necrostatin-1 (Nec-1), an allosteric RIPK1 inhibitor, inhibits necroptosis induced by TNF or the TLR3 ligand poly I:C and abolishes the formation and activation of an RIPK1/3 complex (13–16, 18). Although the molecular details whereby RIPK1 initiates necroptosis are unclear, RIPK3 and the pseudo kinase MLKL appear to be required (2).Genetic studies in mice have revealed cross-regulation between the apoptotic and necroptotic pathways. For example, the FADD/caspase-8/FLICE-like inhibitory protein long form (FLIP_L) complex regulates RIPK1 and RIPK3 activity during development, because the embryonic lethality associated with a caspase-8 deficiency is completely rescued by the absence of RIPK3 (19, 20). Similarly, RIPK1 deficiency rescues FADD-associated embryonic lethality (21). Thus, in the absence of FADD or caspase-8, embryos succumb to RIPK1- and RIPK3-dependent necroptosis. However, Fadd^−/−/Ripk1^−/− mice, die perinatally (21, 22), as do Ripk1^−/− mice, revealing that RIPK1 has prosurvival roles beyond the regulation of the FADD/caspase-8/FLIP_L complex.We have demonstrated that complete RIPK1 deficiency results in increased TNF-induced cell death that can be rescued, in part, by the absence of the TNFR1 (22, 23). However, Ripk1^−/−Tnfr1^−/− animals still succumb (23), indicating that other death ligands/pathways contribute to the RIPK1-associated lethality. Consistent with this hypothesis, RIPK3 deficiency recently has been shown to rescue the perinatal lethality observed in Ripk1^−/−Tnfr1^−/− mice (24, 25). Similarly, combined caspase-8 and RIPK3 deficiency also rescues the RIPK1-associated lethality (24–26). Collectively, these genetic studies in mice reveal that the perinatal death of Ripk1^−/− mice reflects TNF-induced apoptosis and RIPK3-mediated necroptosis. The nature of the ligand(s) or the trigger(s) of RIPK3-mediated necroptosis in vivo remain unclear. However, Ripk1^−/− MEFs are prone to necroptosis induced by poly I:C or by treatment with type I or type II IFN (24, 25), suggesting that these pathways contribute. Although these studies reveal a regulatory role for RIPK1, the multiorgan cell death and inflammation observed in the complete and compound RIPK1-knockout strains have made it difficult to discern the specific tissues that require RIPK1 for survival.     

ZBP1 promotes inflammatory responses downstream of TLR3/TLR4 via timely delivery of RIPK1 to TRIF

ZBP1 is widely recognized as a mediator of cell death for its role in initiating necroptotic, apoptotic, and pyroptotic cell death pathways in response to diverse pathogenic infection. Herein, we characterize an unanticipated role for ZBP1 in promoting inflammatory responses to bacterial lipopolysaccharide (LPS) or double-stranded RNA (dsRNA). In response to both stimuli, ZBP1 promotes the timely delivery of RIPK1 to the Toll-like receptor (TLR)3/4 adaptor TRIF and M1-ubiquitination of RIPK1, which sustains activation of inflammatory signaling cascades downstream of RIPK1. Strikingly, ZBP1-mediated regulation of these pathways is important in vivo, as Zbp1^−/− mice exhibited resistance to LPS-induced septic shock, revealed by prolonged survival and delayed onset of hypothermia due to decreased inflammatory responses and subsequent cell death. Further findings revealed that ZBP1 promotes sustained inflammatory responses by mediating the kinetics of proinflammatory “TRIFosome” complex formation, thus having a profound impact downstream of TLR activation. Given the well-characterized role of ZBP1 as a viral sensor, our results exemplify previously unappreciated crosstalk between the pathways that regulate host responses to bacteria and viruses, with ZBP1 acting as a crucial bridge between the two.

ZBP1 (Z-DNA-binding protein 1) is a cytosolic nucleic acid sensor and RHIM (receptor interacting protein [RIP] homotypic interaction motif) domain–containing protein that has been studied extensively in the context of RIPK3 (RIP kinase)-dependent necroptosis (1–4). As such, ZBP1 uses its two Z-DNA-binding domains to recognize viruses such as murine cytomegalovirus and influenza A virus (IAV) (2, 4–6), driving RHIM-mediated interactions between ZBP1 and RIPK3 and the activation of mixed lineage kinase domain like pseudokinase. This activation cascade ultimately induces necroptosis, as well as the assembly of a RIPK1/Fas receptor-associated death domain protein (FADD)/caspase-8 (CASP8)-containing complex that drives apoptosis in response to viral infection (1). In response to IAV infection, ZBP1 also promotes RIPK3-independent apoptosis, dependent on the direct recruitment of RIPK1 and activation of FADD and CASP8 (1, 7). ZBP1-mediated induction of necroptosis can also be unleashed during mammalian development if RIPK1 is deleted or mutated. Indeed, mutation of the RHIM domain of RIPK1 results in embryonic lethality in mice that can be rescued by the additional deletion of ZBP1 (8, 9).Recently, we reported that when the host inflammatory response is inhibited, ZBP1 initiates CASP8-mediated, RIPK1-dependent pyroptosis in response to bacterial lipopolysaccharide (LPS) (10–12). That is, ZBP1 promoted formation of a prodeath complex (or TRIFosome) downstream of the RHIM domain-containing protein and Toll-like receptor 4 (TLR4) adaptor TRIF (TIR domain containing adaptor protein-inducing interferon [IFN] β), suggesting that ZBP1 might also regulate LPS-induced inflammatory responses downstream of TRIF. Herein, we demonstrate that ZBP1 is important for the core functions of the TLR4 and TLR3 pathways by promoting the production of proinflammatory cytokines in response to LPS or polyinosine-polycytidylic acid (poly(I:C)) in vitro and in vivo. Via RHIM-dependent interactions, ZBP1 tunes the timing and magnitude of the inflammatory response by regulating the kinetics of proinflammatory complex formation and activation of mitogen activated protein kinase (MAPK), nuclear factor-κappa B (NF-κB), and IRF3-mediated signaling cascades. Importantly, deficiency in ZBP1 promotes resistance to LPS-induced septic shock in vivo by damping serum and tissue-specific inflammatory responses and cell death, mirroring the decrease and delay in the inflammatory response observed in vitro in the absence of ZBP1. Together with our previously reported role for ZBP1 in the regulation of the prodeath TRIFosome (10) complex, these data suggest that a similar complex is assembled in the proinflammatory context, thus presenting the TRIFosome as a universal regulator of cell death and inflammatory responses.     

Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, β-galactoside–binding proteins, bind to various gram-negative bacterial LPS, which display β-galactoside–containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1β production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis—a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns that can elicit a host defense response through binding to cell-surface Toll-like receptor 4 (TLR4). Systemic inflammatory response syndrome is induced by overstimulation of the innate immune response via LPSs, resulting in severe multiple organ failure, which is a major cause of death worldwide in intensive care units (1). LPS-induced dimerization of TLR4 initiates signal transduction involving the NF-κB– and MyD88-dependent and -independent pathways, thereby contributing to various inflammatory responses (2). Another set of the immune repertoire, which resides in the cytosol and comprises NLRP1, NLRP3, NAIP/NLRC4, and AIM2, is known as the inflammasome. Inflammasomes can be activated in response to a number of well-defined pathogen-derived ligands and physiological aberrations, which in turn trigger caspase-1–mediated pyroptotic death (3, 4). This process has been associated with strengthening the host defense program to eliminate intracellular bacteria.Recently, a cytosolic LPS-sensing pathway involving caspase-4/5 in humans and caspase-11 in mice was termed the noncanonical inflammasome pathway, and this pathway is independent of TLR4 (5–8). LPSs from extracellular bacteria can enter the cytoplasm and trigger caspase-4/5/11–dependent responses. LPSs can be delivered into the cytosol when LPS-containing outer membrane vesicles (OMVs) from gram-negative bacteria are taken up by the cells or when intracellular bacteria escape from the phagosomes that are damaged by host resistant factors such as guanylate-binding protein and HMGB1 or microbe-derived hemolysins (9–12). LPSs comprise three regions: lipid A, core oligosaccharide, and O-polysaccharide (also termed O-antigen). The lipid A moiety binds directly to the caspase-4/5/11 caspase activation and recruitment domain (CARD, also known as prodomain), leading to caspase oligomerization and activation (7). This event likely mimics the proximity-induced dimerization model of initiator caspase activation (13). Furthermore, caspase-4/5/11 executes downstream signaling events via gasdermin D. Activated inflammatory caspase proteolytically cleaves gasdermin D to create an N-terminal fragment that self-oligomerizes and then inserts into the cell membrane to form pores, causing lytic cell death (14–17). Various stimuli have been identified in the caspase-1–mediated canonical-inflammasome signaling pathway (3, 4), but the detailed mechanism underlying noncanonical inflammasome activation mediated by caspase-4/5/11 remains unclear.Galectins, a family of β-galactoside–binding proteins, can decode host-derived complex glycans and are involved in various biological responses (18–23). Galectins are nucleocytoplasmic proteins synthesized without a classical signal sequence, although they can be secreted through unconventional pathways (19, 21, 23, 24). Recent studies have revealed prominent roles of cytosolic galectins in host defense programs (12, 25, 26). The proposed molecular mechanisms involve the binding of galectins to host glycans exposed to the cytosolic milieu upon endosomal or phagosomal membrane damage. In addition to binding host glycans, galectins also recognize microbial glycans, particularly LPSs (27–30). However, the contribution of galectins to the host response through binding to cytosolic LPSs is unknown.Galectin-3 is an ∼30-kDa protein that contains a carbohydrate-recognition domain (CRD) connected to N-terminal proline, glycine, and tyrosine-rich tandem repeats. Upon binding to multivalent glycoconjugates through its CRD, the protein forms oligomers, which is attributable to the self-association property of its N-terminal region (31, 32). Galectin-3 binds to LPSs of various gram-negative bacteria by recognizing their carbohydrate residues (33–36).Although structural information is scarce (37), existing information suggests that ligand-induced oligomerization of caspase CARD is necessary for the activation of inflammatory caspases (7, 38). Therefore, we hypothesized that galectin-3 may be an intracellular LPS sensor that participates in LPS-induced CARD-mediated inflammatory caspase activation. Specifically, highly ordered arrays of LPS–galectin-3 complexes may amplify caspase-4/5/11 oligomerization and activation. Here, we investigated the formation of galectin-3–LPS–caspase-4/11 complexes in cell-based and cell-free systems. Our findings provide evidence regarding a role of galectin-3 as an intracellular mediator in noncanonical inflammasome activation through LPS glycan recognition.     

Synchronized renal tubular cell death involves ferroptosis

Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia–reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the first-in-class compound ferrostatin-1 (Fer-1). Even in conditions with extraordinarily severe IRI, 16-86 exerts strong protection to an extent which has not previously allowed survival in any murine setting. In addition, 16-86 further potentiates the strong protective effect on IRI mediated by combination therapy with necrostatins and compounds that inhibit mitochondrial permeability transition. Renal tubules thus represent a tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. Finally, ferroptosis mediates postischemic and toxic renal necrosis, which may be therapeutically targeted by ferrostatins and by combination therapy.Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis (1–3). Necroptosis, the best-characterized pathway of regulated necrosis, involves activation of receptor-interacting protein kinase 3 (RIPK3)-mediated phosphorylation of mixed lineage kinase domain-like protein (pMLKL) and subsequent plasma-membrane rupture, which was demonstrated in several disease states, including ischemia–reperfusion injury (IRI) in all organs analyzed (2–6); however, none of these previous studies clearly investigated the mode of cell death in the primary parenchymal cells. Therefore, it remained possible that the protective effects reported upon application of the necroptosis inhibitor necrostatin-1 (Nec-1) and for RIPK3-ko mice involve vascular, nonparenchymal effects. This possibility has been ruled out in non-IRI settings by conditional tissue targeting of proteins involved in the prevention of spontaneously occurring necroptosis, such as RIPK1, and components that regulate its ubiquitinylation status [linear ubiquitinylation chain assembly complex (LUBAC), cellular inhibitors of apoptosis proteins (cIAPs)), caspase-8, and Fas-associated protein with death domain (FADD)] in the gastrointestinal tract (7, 8), the skin (9, 10), the liver (11), and immune cells (12, 13), all of which result in spontaneous RIPK3-mediated tissue necroptosis and inflammation (7–9, 11, 12, 14–17).Ferroptosis is an iron-dependent necrotic type of cell death that occurs due to lipid peroxide accumulation, which routinely is prevented by glutathione peroxidase 4 (GPX4), a glutathione-(GSH)-dependent enzyme, and therefore depends on the functionality of a glu/cys antiporter in the plasma membrane referred to as system X_c-minus (18–20). Ferroptosis has been reported to cause several diseases and may be interfered with in vitro by the small molecule ferrostatin-1 (Fer-1) (18); however, Fer-1 was suggested to have low in vivo functionality due to potential metabolic and plasma instability.In the present studies, we used inducible, conditional kidney tubule-specific genetic deletion of FADD and caspase-8, intravital microscopy, fresh isolation of primary kidney tubules, and four preclinical models of acute organ failure to further assess the relative roles of necroptosis and ferroptosis. We find that ferroptosis is of functional in vivo relevance in acute tubular necrosis and IRI, and we introduce, to our knowledge, the first ferroptosis inhibitor that is applicable for inhibition of ferroptosis in vivo. We conclude that specific combinatory therapies will be most promising for the prevention of clinically relevant IRI and that the nephron represents, to our knowledge, the first described tissue that is not sensitized to necroptosis by loss of FADD or caspase-8.     

Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens

Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1–independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.Pattern recognition receptors (PRRs) of the innate immune system are critical for promoting defense against bacterial pathogens (1). Cytosolic PRRs are key for discriminating between pathogenic and nonpathogenic bacteria, because many pathogens access the host cytosol, a compartment where microbial products are typically not found (2). Cytosolic PRRs respond to patterns of pathogenesis that are often associated with virulent bacteria, such as the use of pore-forming toxins or injection of effector molecules through specialized secretion systems (3). A subset of cytosolic PRRs induces the formation of multiprotein complexes known as inflammasomes (4). In mice, the canonical inflammasome activates caspase-1, an inflammatory caspase that mediates cell death and IL-1 family cytokine secretion (5, 6). Additionally, the noncanonical inflammasome activates caspase-11 in response to many gram-negative bacteria (7–14). The canonical and noncanonical inflammasomes differentially regulate release of IL-1α and IL-1β (7). Caspase-11 mediates LPS-induced septic shock in mice (7, 15), and caspase-11 responds to cytoplasmic LPS independent of Toll-like receptor 4 (16, 17).In addition to its pathologic role in septic shock, the noncanonical inflammasome is critical for host defense in mice (11, 18). However, in humans, it is unclear whether an analogous noncanonical inflammasome exists. Whereas mice encode caspase-11, humans encode two putative functional orthologs: caspase-4 and caspase-5 (19–21). All three inflammatory caspases bind directly to LPS in vitro (22). In murine macrophages, caspase-1 and caspase-11 have both distinct and overlapping roles in the release of IL-1α and IL-1β and the induction of cell death (7). However, the precise role of the human inflammatory caspases in the context of infection by bacterial pathogens remains unclear.To elucidate how human inflammasome activation is regulated, we investigated the contribution of inflammatory caspases to the response against gram-negative bacterial pathogens in human macrophages. Here, we show that both canonical caspase-1–dependent and noncanonical caspase-1–independent inflammasomes are activated in primary human macrophages and that caspase-4 mediates caspase-1–independent inflammasome responses against several bacterial pathogens, including Legionella pneumophila, Yersinia pseudotuberculosis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Importantly, noncanonical inflammasome activation in human macrophages is specific for virulent strains of these bacteria that translocate bacterial products into the host cytosol via the virulence-associated type III secretion system (T3SS) or type IV secretion system (T4SS). Thus, caspase-4 is critical for noncanonical inflammasome responses against virulent gram-negative bacteria in human macrophages.     

Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8–mediated apoptosis of macrophages to promote lethal anthrax

Lethal toxin (LeTx)-mediated killing of myeloid cells is essential for Bacillus anthracis, the causative agent of anthrax, to establish systemic infection and induce lethal anthrax. The “LeTx-sensitive” NLRP1b inflammasome of BALB/c and 129S macrophages swiftly responds to LeTx intoxication with pyroptosis and secretion of interleukin (IL)-1β. However, human NLRP1 is nonresponsive to LeTx, prompting us to investigate B. anthracis host–pathogen interactions in C57BL/6J (B6) macrophages and mice that also lack a LeTx-sensitive Nlrp1b allele. Unexpectedly, we found that LeTx intoxication and live B. anthracis infection of B6 macrophages elicited robust secretion of IL-1β, which critically relied on the NLRP3 inflammasome. TNF signaling through both TNF receptor 1 (TNF-R1) and TNF-R2 were required for B. anthracis-induced NLRP3 inflammasome activation, which was further controlled by RIPK1 kinase activity and LeTx-mediated proteolytic inactivation of MAP kinase signaling. In addition to activating the NLRP3 inflammasome, LeTx-induced MAPKK inactivation and TNF production sensitized B. anthracis-infected macrophages to robust RIPK1- and caspase-8–dependent apoptosis. In agreement, purified LeTx triggered RIPK1 kinase activity- and caspase-8–dependent apoptosis only in macrophages primed with TNF or following engagement of TRIF-dependent Toll-like receptors. Consistently, genetic and pharmacological inhibition of RIPK1 inhibited NLRP3 inflammasome activation and apoptosis of LeTx-intoxicated and B. anthracis-infected macrophages. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the in vivo pathophysiological relevance of this cytotoxic mechanism. Collectively, these results establish TNF- and RIPK1 kinase activity–dependent NLRP3 inflammasome activation and macrophage apoptosis as key host–pathogen mechanisms in lethal anthrax.

The bacterial pathogen Bacillus anthracis is a rare, but notoriously deadly pathogen in humans with mortality rates from anthrax varying from ∼20% for cutaneous anthrax to 80% and higher for inhalation anthrax. This encapsulated, spore-forming, gram-positive bacterial pathogen efficiently kills infected hosts through the systemic action of two secreted toxins (1). Edema toxin (EdTx) and lethal toxin (LeTx) share a receptor-binding protein named protective antigen (PA) that transfers the edema factor (EF) and lethal factor (LF) moieties into the cytosol of target cells, where the latter exert their cytopathic and cytotoxic effects (1, 2).Studies in macaques and mice identified LeTx as a major virulence factor driving systemic dispersion of vegetative bacteria, which ultimately may result in fatal anthrax (3, 4). LeTx internalization by macrophages drives macrophage cell death, which is a key early pathogenic event during spore infections that allows vegetative bacteria to establish systemic infection of its host (5). LF is a highly selective Zn²⁺-dependent metalloprotease that, once internalized, cleaves a subset of mitogen-activated protein kinase kinases (MAPKKs) to abolish downstream MAPK signaling in LeTx-intoxicated macrophages (6). In addition, macrophages of BALB/c and 129S mice express a LeTx-sensitive Nlrp1b allele that responds to LF-mediated cleavage with NLRP1b inflammasome activation and pyroptosis (7–9). However, human NLRP1 and the Nlrp1b allele of C57BL/6J (B6) macrophages are nonresponsive to LeTx, suggesting that B. anthracis may induce macrophage cell death through alternative mechanisms that are poorly understood.Here, we show that B. anthracis infection induces NLRP3 inflammasome activation and caspase-8–mediated apoptosis of B6 macrophages. Notably, B. anthracis sensitizes macrophages by promoting TNF production concomitantly with LeTx-mediated inactivation of p38 MAPK signaling. LeTx intoxication of TLR3/4- or TNF-activated macrophages similarly sensitized macrophages to TNF- and RIPK1 kinase activity–dependent NLRP3 inflammasome activation and cell death induction. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the in vivo pathophysiological relevance of this cytotoxic mechanism in lethal anthrax.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex

Pathogen recognition by nucleotide-binding oligomerization domain-like receptor (NLR) results in the formation of a macromolecular protein complex (inflammasome) that drives protective inflammatory responses in the host. It is thought that the number of inflammasome complexes forming in a cell is determined by the number of NLRs being activated, with each NLR initiating its own inflammasome assembly independent of one another; however, we show here that the important foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) simultaneously activates at least two NLRs, whereas only a single inflammasome complex is formed in a macrophage. Both nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 are simultaneously present in the same inflammasome, where both NLRs are required to drive IL-1β processing within the Salmonella-infected cell and to regulate the bacterial burden in mice. Superresolution imaging of Salmonella-infected macrophages revealed a macromolecular complex with an outer ring of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and an inner ring of NLRs, with active caspase effectors containing the pro–IL-1β substrate localized internal to the ring structure. Our data reveal the spatial localization of different components of the inflammasome and how different members of the NLR family cooperate to drive robust IL-1β processing during Salmonella infection.Inflammasomes are cytosolic multimeric protein complexes formed in the host cell in response to the detection of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Formation of the inflammasome in response to PAMPs is critical for host defense because it facilitates processing of the proinflammatory cytokines pro–IL-1β and pro–IL-18 into their mature forms (1). The inflammasome also initiates host cell death in the form of pyroptosis, releasing macrophage-resident microbes to be killed by other immune mechanisms (2). The current paradigm is that there are individual, receptor-specific inflammasomes consisting of one nucleotide-binding oligomerization domain-like receptor (NLR; leucine-rich repeat–containing) or PYHIN [pyrin domain and hematopoietic expression, interferon-inducible nature, and nuclear localization (HIN) domain-containing] receptor, the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD; ASC), and caspase-1 (3). How the protein constituents of the inflammasome are spatially orientated is unclear.Nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 (NLRC4) and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 (NLRP3) are the best-characterized inflammasomes, especially with respect to their responses to pathogenic bacteria. The NLRC4 inflammasome is activated primarily by bacteria, including Aeromonas veronii (4), Escherichia coli (5), Listeria monocytogenes (6, 7), Pseudomonas aeruginosa (5), Salmonella enterica serovar Typhimurium (S. Typhimurium) (5, 8–10), and Yersinia species (11). In mouse macrophages, the NLRC4 inflammasome responds to flagellin and type III secretion system-associated needle or rod proteins (5, 8, 9) after their detection by NLR family, apoptosis inhibitory protein (NAIP) 5 or NAIP6 and NAIP1 or NAIP2, respectively (12–15). Phosphorylation of NLRC4 at a single, evolutionarily conserved residue, Ser 533, by PKCδ kinase is required for NLRC4 inflammasome assembly (16). The NLRP3 inflammasome is activated by a large repertoire of DAMPs, including ATP, nigericin, maitotoxin, uric acid crystals, silica, aluminum hydroxide, and muramyl dipeptide (17–20). NLRP3 is also activated by bacterial PAMPs from many species, including Aeromonas species (4, 21), L. monocytogenes (6, 7, 22), Neisseria gonorrhoeae (23), S. Typhimurium (10), Streptococcus pneumoniae (24), and Yersinia species (11). The mechanisms by which NLRC4 and NLRP3 inflammasomes contribute to host defense against bacterial pathogens are emerging; however, little is known about the dynamics governing inflammasome assembly in infections caused by bacteria that activate multiple NLRs, such as S. Typhimurium (10), A. veronii (4), and Yersinia (11).NLRP3 does not have a CARD and requires ASC to interact with the CARD of procaspase-1. This interaction requires a charged interface around Asp27 of the procaspase-1 CARD (25). Whether ASC is also required for the assembly of the NLRC4 inflammasome is less clear. NLRC4 contains a CARD that can interact directly with the CARD of procaspase-1 (26); however, ASC is required for some of the responses driven by NLRC4 (27). Macrophages infected with S. Typhimurium or other pathogens exhibit formation of a distinct cytoplasmic ASC focus or speck, which can be visualized under the microscope and is indicative of inflammasome activation (10, 28, 29). Our laboratory and others have shown that only one ASC speck is formed per cell irrespective of the stimulus used (29–32). However, many bacteria activate two or more NLRs, and it is unclear whether a singular inflammasome is formed at a time or if multiple inflammasomes are formed independent of each other, with each inflammasome containing one member of the NLR family.In this study, we describe the endogenous molecular constituents of the Salmonella-induced inflammasome and their spatial orientation. In cross-section, ASC forms a large external ring with the NLRs and caspases located internally. Critically, NLRC4, NLRP3, caspase-1, and caspase-8 coexist in the same ASC speck to coordinate pro–IL-1β processing. All ASC specks observed contained both NLRC4 and NLRP3. These results suggest that Salmonella infection induces a single inflammasome protein complex containing different NLRs and recruiting multiple caspases to coordinate a multifaceted inflammatory response to infection.     

Cytosolic flagellin-induced lysosomal pathway regulates inflammasome-dependent and -independent macrophage responses

NAIP5/NLRC4 (neuronal apoptosis inhibitory protein 5/nucleotide oligomerization domain-like receptor family, caspase activation recruitment domain domain-containing 4) inflammasome activation by cytosolic flagellin results in caspase-1–mediated processing and secretion of IL-1β/IL-18 and pyroptosis, an inflammatory cell death pathway. Here, we found that although NLRC4, ASC, and caspase-1 are required for IL-1β secretion in response to cytosolic flagellin, cell death, nevertheless, occurs in the absence of these molecules. Cytosolic flagellin-induced inflammasome-independent cell death is accompanied by IL-1α secretion and is temporally correlated with the restriction of Salmonella Typhimurium infection. Despite displaying some apoptotic features, this peculiar form of cell death do not require caspase activation but is regulated by a lysosomal pathway, in which cathepsin B and cathepsin D play redundant roles. Moreover, cathepsin B contributes to NAIP5/NLRC4 inflammasome-induced pyroptosis and IL-1α and IL-1β production in response to cytosolic flagellin. Together, our data describe a pathway induced by cytosolic flagellin that induces a peculiar form of cell death and regulates inflammasome-mediated effector mechanisms of macrophages.Flagellin, the monomeric subunit of flagella present in Gram-negative and Gram-positive bacteria, is one of the few protein structures that can activate both transmembrane and cytosolic pattern recognition receptors of the innate immune system. Extracellular flagellin is recognized by the transmembrane Toll-like receptor (TLR)5 (1). On the other hand, flagellin can be directly delivered into the cytosol by transport systems, such as the type III secretion system (T3SS) of Salmonella (2) and the type IV secretion system (T4SS) of Legionella (3). Once in the cytosol, flagellin is sensed by the inflammasome complex comprised of the NOD-like receptor (NLR) proteins neuronal apoptosis inhibitory protein (NAIP)5 and NLRC4 [NLR family, caspase activation recruitment domain (CARD) domain-containing 4] (2–5).Both TLR5 and NAIP5/NLRC4 receptors recognize conserved regions of flagellin. TLR5 is thought to detect a region of flagellin located in the D1 domain (6), whereas a sequence of three leucine residues that is present in the C-terminal D0 domain of flagellin is required to activate the NAIP5/NLRC4 inflammasome (7). Despite some redundant roles that are attributed to NLRC4 and NAIP5 in flagellin-mediated macrophage activation (7), a new model for NAIP5/NLRC4 inflammasome activation in response to flagellin was recently proposed (8, 9). In this model, NAIP5 acts as an immune sensor protein that specifically binds to flagellin (9). The interaction between NAIP5 and flagellin promotes the recruitment of NLRC4 through the NOD domain. The formation of this protein complex leads to the association of NLRC4 with procaspase-1 via CARD-CARD interactions. Additionally, NLRC4 can recruit the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which also contains a CARD domain and is able to recruit and process procaspase-1.Caspase-1 activation results in the cleavage and secretion of biologically active forms of the inflammatory cytokines interleukin (IL)-1β and IL-18 (10) and the induction of a form of cell death named pyroptosis (11). The activation of caspase-1 in response to cytosolic flagellin by the NAIP5/NLRC4 inflammasome complex can also induce other effector mechanisms to restrict infections, such as caspase-7–dependent phagosome maturation (4, 12) and the activation of inducible nitric oxide synthase (iNOS) by macrophages (13). Both of these effector mechanisms lead to the inhibition of Legionella pneumophila replication. Importantly, caspase-1–induced IL-1β and IL-18 are not involved in phagosome maturation (4, 12), induction of pyroptosis (14), or iNOS activation (13), suggesting that caspase-1 mediates independent effects that cooperate to clear infections.Although the NAIP5/NLRC4 inflammasome complex is involved in the control of many bacterial infections, such as infection with Salmonella Typhimurium (2, 5), Shigella flexneri (15), Pseudomonas aeruginosa (16, 17), L. pneumophila (3, 4), and Listeria monocytogenes (18), the precise effector mechanism mediated by these receptors is not completely understood. Among the NAIP5/NLRC4 inflammasome-mediated effector mechanisms that have been implicated with intracellular bacterial replication control, pyroptosis has received great attention.Pyroptosis has been described as a programmed cell death pathway that uniquely depends on caspase-1 (19). Recently, it was demonstrated that the enteric pathogenic bacteria Escherichia coli, Citrobacter rodentium, and Vibrio cholerae and the cholera toxin B subunit can trigger the activation of a noncanonical inflammasome that targets caspase-11 (also known as caspase-4 in humans and related to caspase-1) (20). These stimuli induce cell death in a caspase-11–dependent fashion, but the process is not dependent on ASC, NLRC4, or caspase-1. Interestingly, this process of cell death (also named pyroptosis) is accompanied by the secretion of IL-1α but not by the secretion of IL-1β (which requires caspase-1). Importantly, the 129 mouse strain that was used to generate the first caspase-1^−/− mutants (21, 22) harbors a mutation in the caspase-11 locus that impairs caspase-11 function. Because of the close proximity in the genome between the caspase-1 and caspase-11 genes, the two proteins cannot be segregated by recombination. Therefore, these caspase-1^−/− mice are also defective for caspase-11 (20).Importantly, although pyroptosis is regulated by caspase activation, similarly to apoptosis, inhibition of or genetic deficiency in apoptotic caspase does not rescue cells from pyroptosis (11, 23). In addition, pyroptosis and apoptosis provide distinct outcomes for the immune response, which may be explained by the different morphological and biochemical changes that are observed in cells undergoing these forms of cell death (24, 25). Activation of caspase-1/11 results in the rapid formation of pores in the plasma membrane that dissipate cellular ionic gradients. This process allows the influx of water into the cells, resulting in cell swelling, osmotic lysis, and the release of intracellular contents (25, 26). The loss of plasma membrane integrity and the secretion of inflammatory mediators during pyroptosis, including IL-1β and IL-18, results in the induction of a strong inflammatory response (27). The inflammatory milieu produced by pyroptosis could result in the recruitment of effector cells to the site of infection as a mechanism of pathogen clearance. Recently, it was demonstrated that the ectopic expression of the Salmonella flagellin protein FliC during the intracellular phase of infection triggers pyroptosis of infected cells in vivo (14). The bacteria released by the pyroptotic macrophages were controlled by infiltrating neutrophils through a reactive oxygen species-dependent mechanism.Despite the evidence implicating pyroptosis as an important host defense mechanism to clear intracellular pathogens, the molecular regulation of pyroptosis is poorly understood. Here, we analyzed the regulation of macrophage death using purified flagellin as a single, death-inducing stimulus. Our data demonstrate that cytosolic flagellin is able to induce cell death in the absence of caspase-1/11. Although displaying some apoptotic features, such as cell shrinkage and the formation of membrane blebs, cytosolic flagellin-induced caspase-1/11–independent cell death does not require apoptotic caspases but depends on lysosomal events. Similar to pyroptosis, cytosolic flagellin-induced caspase-1/11–independent cell death results in the release of intracellular inflammatory contents. Caspase-1/11–independent cell death also contributes to the control of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection by macrophages, supporting the existence of an effector mechanism important to restrict bacterial infection. Finally, our data provide evidences that lysosomal cathepsins also regulate IL-1β secretion and pyroptosis in response to cytosolic flagellin. Taken together, our results suggests lysosome events as a central regulator of both inflammasome-dependent and inflammasome-independent macrophage responses induced by cytosolic flagellin.     

Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function

Inflammasomes are caspase-1–activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11^−/− cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.Anthrax is a zoonotic disease caused by the Gram-positive bacterium Bacillus anthracis. B. anthracis provokes a shock-like syndrome that can prove fatal to the host (1) and has recently gained notoriety as a potential bioterrorism agent. Anthrax pathogenicity relies on its ability to secrete three virulence proteins, which combine with each other to form two toxins. The protective antigen (PA) combines with the edema factor (EF) to form the edema toxin (2, 3). EF is an adenylate cyclase that causes edema of the infected tissue. The binary combination of PA with lethal factor (LF) gives rise to the most virulent factor, called lethal toxin (LT), responsible for the systemic symptoms and death of the infected animal. To escape the host immune response, LT impairs the host innate immunity by killing macrophages (4–6). The PA protein interacts with LF and binds to cell surface receptors, enabling endocytosis of the LT complex. In the acidic compartment, PA forms pores allowing the delivery of LF to the cytosol. LF is a zinc metalloprotease that was shown to cleave the N-terminal region of many MAP kinase kinases and to induce apoptosis of macrophages. LT also triggers pyroptosis through the formation of a caspase-1–activating platform, named “inflammasome” (6–8).Inflammasomes are multiprotein complexes of the innate immune response that control caspase-1 activity and pro–IL-1β and pro–IL-18 maturation. Most inflammasomes are composed of specific cytosolic pathogen recognition receptors (PRRs), as well as the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC) adaptor protein that enables the recruitment and activation of the caspase-1 protease. Once caspase-1 is oligomerized within an inflammasome platform, the enzyme undergoes autoproteolysis to form heterodimers of active caspase-1 (9–12). In the mouse, at least five distinct inflammasomes have been described, distinguished by the PRR that induces the complex formation. The PRRs capable of participating in inflammasome platform formation are either members of the nod-like receptor (NLR) family (e.g., NLRP1, NLRP3, or NLRC4) or of the PYrin and HIN (PYHIN) family (e.g., AIM2) (13, 14). ASC is composed of a pyrin domain (PYD) and a caspase activation and recruitment domain (CARD). ASC interacts with a PYD-containing PRR via its PYD domain and recruits the CARD domain of caspase-1 via its CARD domain. Thus, ASC is essential to the formation of the inflammasome by receptors such as NLRP3 or AIM2 (15–18). However, its presence is dispensable for NLRC4, which contains a CARD in place of a PYD, allowing direct interaction with the CARD domain of caspase-1 (19, 20).Past studies have determined that certain mouse strains are more sensitive than others to LT cytotoxicity, and genetic studies identified NLRP1b as the factor conferring mouse strain susceptibility to anthrax LT (21). The mouse genome contains three different NLRP1 isoforms (a, b, and c) and a functional NLRP1b was found to be expressed by the mouse strains sensitive to LT (e.g., BALB/c or 129 background). Expression of NLRP1b was shown to mediate IL-1β release and caspase-1–mediated cell death in response to LT (7, 21, 22). Mouse NLRP1b differs structurally from human NLRP1 in that it lacks the N-terminal PYD (23). The absence of the PYD suggests that NLRP1b can directly engage caspase-1 without a requirement for ASC. However, studies dissecting the mechanism of NLRC4 inflammasome activation demonstrated that ASC is required for the amplification of caspase-1 autoprocessing and IL-1β secretion but not for pyroptosis (19, 20). Cell lysis mediated by LT was shown to be dependent on sodium and potassium fluxes (24), and high extracellular potassium inhibited IL-1β secretion upon LT treatment, suggesting a role for the NLRP3 inflammasome in LT sensing (22, 25). Therefore, we investigated whether NLRP3 and/or ASC were required for caspase-1 activation in response to LT. The NLRP3, ASC, and caspase-1 mouse knockout strains were backcrossed into the BALB/c background and the response of macrophages and dendritic cells (DCs) to LT intoxication was studied. Our data reveal that (i) in response to LT, ASC is dispensable for caspase-1 activation, but uncleavable caspase-1 is fully active; and (ii) upon activation of the NLRP3 inflammasome, uncleavable caspase-1 is inactive.     

Precise spatial structure impacts antimicrobial susceptibility of S. aureus in polymicrobial wound infections

A hallmark of microbial ecology is that interactions between members of a community shape community function. This includes microbial communities in human infections, such as chronic wounds, where interactions can result in more severe diseases. Staphylococcus aureus is the most common organism isolated from human chronic wound infections and has been shown to have both cooperative and competitive interactions with Pseudomonas aeruginosa. Still, despite considerable study, most interactions between these microbes have been characterized using in vitro well-mixed systems, which do not recapitulate the infection environment. Here, we characterized interactions between S. aureus and P. aeruginosa in chronic murine wounds, focusing on the role that both macro- and micro-scale spatial structures play in disease. We discovered that S. aureus and P. aeruginosa coexist at high cell densities in murine wounds. High-resolution imaging revealed that these microbes establish a patchy distribution, only occupying 5 to 25% of the wound volume. Using a quantitative framework, we identified a precise spatial structure at both the macro (mm)- and micro (µm)-scales, which was largely mediated by P. aeruginosa production of the antimicrobial 2-heptyl-4-hydroxyquinoline N-oxide, while the antimicrobial pyocyanin had no impact. Finally, we discovered that this precise spatial structure enhances S. aureus tolerance to aminoglycoside antibiotics but not vancomycin. Our results provide mechanistic insights into the biogeography of S. aureus and P. aeruginosa coinfected wounds and implicate spatial structure as a key determinant of antimicrobial tolerance in wound infections.

Polymicrobial human infections are a major burden on human health. These infections are often more tolerant to antibiotics and have worse clinical outcomes compared to their single-microbe counterparts (1–7). Properties specific to polymicrobial infections are often attributed to interactions occurring between microbes, and much work has been done to identify and mechanistically understand these interactions (8–12). Recent evidence using preclinical infection models has shown that interactions between microbes impact the micron-scale spatial structure of the infecting community (13–16), implicating the spatial structure as a key component controlling community function, and thus infection outcomes (17). However, most of our understanding of polymicrobial interactions is derived from studies using in vitro models (13, 14). Hence, key elements of infection dynamics and the role of host factors are often overlooked.Pseudomonas aeruginosa and Staphylococcus aureus are commonly used to study microbe–microbe interactions, both in vitro and in vivo (11, 13, 18–24). These microbes cooccur in several polymicrobial human infections, including chronic wounds and in the lungs of people with cystic fibrosis (1–3, 5, 25–29). There is conflicting evidence regarding the impact of coinfection on human disease outcomes, with some studies concluding that P. aeruginosa alone has worse outcomes (32–34) while others conclude that P. aeruginosa–S. aureus coinfections lead to more severe diseases (35–37). The experimental data are clearer in murine models of infection, which have shown that coinfection can result in increased antibiotic tolerance and worse infection outcomes (8, 10, 20, 28). While the mechanisms controlling these synergistic interactions are largely unknown in vivo, it has been hypothesized that P. aeruginosa and S. aureus occupy distinct regions in human chronic wounds (28), suggesting that biogeography may play a role in mediating polymicrobial wound infection outcomes.Here, we collected more than 100 high-resolution confocal images of mouse chronic wounds infected with P. aeruginosa and S. aureus in mono- and co-infection. Using these images, we quantified the 3-dimensional macro- and micron-scale spatial structure of P. aeruginosa and S. aureus communities in vivo and defined the role of known P. aeruginosa extracellular antimicrobials on the spatial structure. We discovered that S. aureus and P. aeruginosa coexist in mouse wound infections at high bacterial densities, but their distribution is patchy. In addition, we discovered and quantified a precise, micron-scale spatial structure dependent on the P. aeruginosa-secreted small-molecule 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and that this spatial structure is different at the healing edge versus the center of the wound. Finally, we show that the community spatial structure has clinically important outcomes, including altered antibiotic tolerance.     

Reciprocal antagonistic regulation of E3 ligases controls ACC synthase stability and responses to stress

Ethylene influences plant growth, development, and stress responses via crosstalk with other phytohormones; however, the underlying molecular mechanisms are still unclear. Here, we describe a mechanistic link between the brassinosteroid (BR) and ethylene biosynthesis, which regulates cellular protein homeostasis and stress responses. We demonstrate that as a scaffold, 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS), a rate-limiting enzyme in ethylene biosynthesis, promote the interaction between Seven-in-Absentia of Arabidopsis (SINAT), a RING-domain containing E3 ligase involved in stress response, and ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-like (EOL) proteins, the E3 ligase adaptors that target a subset of ACS isoforms. Each E3 ligase promotes the degradation of the other, and this reciprocally antagonistic interaction affects the protein stability of ACS. Furthermore, 14–3-3, a phosphoprotein-binding protein, interacts with SINAT in a BR-dependent manner, thus activating reciprocal degradation. Disrupted reciprocal degradation between the E3 ligases compromises the survival of plants in carbon-deficient conditions. Our study reveals a mechanism by which plants respond to stress by modulating the homeostasis of ACS and its cognate E3 ligases.

Protein homeostasis is a core mechanism for maintaining cellular function, which enables organisms to rapidly respond to environmental stress in a specific manner. The ubiquitin–proteasome-mediated degradation pathway is one of the main pathways that govern protein homeostasis in cells and has been linked to diverse functions in plants, including hormone signaling, plant defense response, photomorphogenesis, and stress response (1–6).The function of the gaseous hormone ethylene is largely regulated by the ubiquitin–proteasome system (7). In ethylene signaling, the abundance of positive regulators ETHYLENE-INSENSITIVE 2 (EIN2) and EIN3 is regulated by E3 ubiquitin ligases, EIN2-TARGETING PROTEIN 1 (ETP1) and ETP2 or EIN3-BINDING F-BOX PROTEIN 1 (EBF1) and EBF2, respectively (3, 8). Similar to the signaling pathway, in ethylene biosynthesis, the abundance of a subset of 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS), the rate-limiting enzymes in the pathway, is specifically regulated by ETHYLENE OVERPRODUCER 1 (ETO1) and its two paralogs, ETO1-like 1 (EOL1) and EOL2. ETO1/EOLs are components of a CULLIN3 E3 ligase, which specifically recognize type-2 ACS isoforms for rapid degradation via the 26S proteasome (9, 10). E3 ligase substrate-specificity subunits such as ETO1/EOLs determine the accessibility of E3 ligase complex to the substrate; thus, the abundance of ETO1/EOLs is an important regulatory factor for determining ACS stability in plants (11). The stability of EOL2 has been shown to be negatively regulated by 14–3-3, a family of phosphoprotein-binding proteins, though the underlying mechanism remains elusive (12).Arabidopsis contains eight functional ACS isoforms that can be grouped into three types, namely type-1 (ACS2, 6), type-2 (ACS4, 5, 8, 9, and 11), and type-3 (ACS7), based on the presence or absence of phosphorylation sites in the C-terminal domains (9, 13, 14). The stability of different ACS is differentially regulated by diverse stimuli, including phytohormones (14–16). Most plant hormones regulate the protein stability of ACS with distinct effects on different ACS isoforms (15, 16). Brassinosteroid (BR) is one such hormone that regulates the protein stability of ACS. However, the underlying mechanisms and molecular components involved in the process are unknown (15). A component that likely plays a role in BR–ethylene crosstalk is the 14–3-3 proteins. The 14–3-3 proteins are evolutionally well-conserved dimeric proteins in all eukaryotic organisms and are involved in varied biological processes via phosphorylation-dependent protein–protein interactions (17–19). The Arabidopsis 14–3-3 family consists of 13 isoforms, and their roles have been suggested in a diverse range of physiological processes including BR signaling (20, 21), ethylene biosynthesis (12, 22), abiotic stress response (22–24), and light signaling (25). In the BR signaling pathway, 14–3-3 proteins interact with multiple BR signaling molecules, including BRASSINOID INSENSITIVE 1 (BRI1), BRI1 KINASE INHIBITOR 1 (BKI1), and BRI1-EMS SUPPRESSOR 1 (BES1)/BRASSINAZOLE-RESISTANT 1 (BZR1), resulting in the regulation of the BR signaling pathway (17). The role of 14–3-3 proteins has been implicated in ethylene biosynthesis via their interaction with ACS in rice, barley, and Arabidopsis (26–29). In Arabidopsis, 14–3-3 positively regulates the protein stability of ACS5 through increased turnover of EOL2 or through an ETO1/EOLs-independent mechanism (12); however, the detailed mechanism such as the stimuli triggering the 14–3-3–mediated regulation of ACS and ETO1/EOL stability or other regulatory components in the process remains unknown. Given the roles of 14–3-3 in ethylene biosynthesis and BR signaling, 14–3-3 could be a crosstalk point that integrates the interaction between ethylene biosynthesis and BR signaling in a phosphorylation-dependent manner.SEVEN-IN-ABSENTIA (SINA) is a RING-type E3 ligase that has been linked to protein degradation and stress response in Drosophila, Mammalian, and plants (30–36). Arabidopsis contains a family of five SINA of Arabidopsis (SINAT) genes that encode two distinct clades of SINAT proteins (SINAT1 and 2; SINAT3, 4, and 5) based on sequence similarities (23). Several recent studies in different plant species have demonstrated that SINA family members play a role in response to abiotic and biotic stresses, including cold, drought, and pathogen invasion; some of these are linked to abscisic acid (ABA) or BR hormone signaling and autophagy, a highly conserved cellular degradation process linked to stress response (23, 33, 37). An autophagy receptor, DOMINANT SUPPRESSOR OF KAR 2 (DSK2), controls the degradation of BES1, a positive regulator for BR signaling, and SINAT2 participates in targeting BES1 for degradation via the DSK2 autophagy receptor under drought and fixed carbon starvation (33). SINAT E3 ligases also regulate the activity and/or stability of a subset of AUTOPHAGY-RELATED PROTEINS (ATGs) (30, 38). They also regulate the stability of FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), the endosomal sorting complex required for the transport (ESCRT) component, thus controlling autophagy (30, 39–41). Intriguingly, SINAT2 has been identified as a putative 14–3-3–interacting protein along with EOL2 and several ACS isoforms through the proteomic profiling of purified complexes from Arabidopsis (27).In this study, we investigated the regulatory mechanism for ACS5 protein stability through SINAT E3 ligases. Strikingly, ACS5 acts as a scaffold that tethers SINAT2 and EOL2 in a functional complex, increasing the stability of ACS5 via the reciprocal degradation of SINAT2 and EOL2. 14–3-3 activates the reciprocal degradation of SINAT2 and EOL2 through direct interaction with SINAT2 only in the presence of BR, thereby linking ethylene biosynthesis and BR signaling. Our results reveal a regulatory mechanism that allows the simultaneous fine-tuning of the protein abundance of ACS and its cognate E3 ligases, which is critical for stress response via autophagy.     

Microscopic origins of the crystallographically preferred growth in evaporation-induced colloidal crystals

Unlike crystalline atomic and ionic solids, texture development due to crystallographically preferred growth in colloidal crystals is less studied. Here we investigate the underlying mechanisms of the texture evolution in an evaporation-induced colloidal assembly process through experiments, modeling, and theoretical analysis. In this widely used approach to obtain large-area colloidal crystals, the colloidal particles are driven to the meniscus via the evaporation of a solvent or matrix precursor solution where they close-pack to form a face-centered cubic colloidal assembly. Via two-dimensional large-area crystallographic mapping, we show that the initial crystal orientation is dominated by the interaction of particles with the meniscus, resulting in the expected coalignment of the close-packed direction with the local meniscus geometry. By combining with crystal structure analysis at a single-particle level, we further reveal that, at the later stage of self-assembly, however, the colloidal crystal undergoes a gradual rotation facilitated by geometrically necessary dislocations (GNDs) and achieves a large-area uniform crystallographic orientation with the close-packed direction perpendicular to the meniscus and parallel to the growth direction. Classical slip analysis, finite element-based mechanical simulation, computational colloidal assembly modeling, and continuum theory unequivocally show that these GNDs result from the tensile stress field along the meniscus direction due to the constrained shrinkage of the colloidal crystal during drying. The generation of GNDs with specific slip systems within individual grains leads to crystallographic rotation to accommodate the mechanical stress. The mechanistic understanding reported here can be utilized to control crystallographic features of colloidal assemblies, and may provide further insights into crystallographically preferred growth in synthetic, biological, and geological crystals.

As an analogy to atomic crystals, colloidal crystals are highly ordered structures formed by colloidal particles with sizes ranging from 100 nm to several micrometers (1–6). In addition to engineering applications such as photonics, sensing, and catalysis (4, 5, 7, 8), colloidal crystals have also been used as model systems to study some fundamental processes in statistical mechanics and mechanical behavior of crystalline solids (9–14). Depending on the nature of interparticle interactions, many equilibrium and nonequilibrium colloidal self-assembly processes have been explored and developed (1, 4). Among them, the evaporation-induced colloidal self-assembly presents a number of advantages, such as large-size fabrication, versatility, and cost and time efficiency (3–5, 15–18). In a typical synthesis where a substrate is immersed vertically or at an angle into a colloidal suspension, the colloidal particles are driven to the meniscus by the evaporation-induced fluid flow and subsequently self-assemble to form a colloidal crystal with the face-centered cubic (fcc) lattice structure and the close-packed {111} plane parallel to the substrate (2, 3, 19–23) (see Fig. 1A for a schematic diagram of the synthetic setup).Open in a separate windowFig. 1.Evaporation-induced coassembly of colloidal crystals. (A) Schematic diagram of the evaporation-induced colloidal coassembly process. “G”, “M”, and “N” refer to “growth,” “meniscus,” and “normal” directions, respectively. The reaction solution contains silica matrix precursor (tetraethyl orthosilicate, TEOS) in addition to colloids. (B) Schematic diagram of the crystallographic system and orientations used in this work. (C and D) Optical image (Top Left) and scanning electron micrograph (SEM) (Bottom Left) of a typical large-area colloidal crystal film before (C) and after (D) calcination. (Right) SEM images of select areas (yellow rectangles) at different magnifications. Corresponding fast-Fourier transform (see Inset in Middle in C) shows the single-crystalline nature of the assembled structure. (E) The 3D reconstruction of the colloidal crystal (left) based on FIB tomography data and (right) after particle detection. (F) Top-view SEM image of the colloidal crystal with crystallographic orientations indicated.While previous research has focused on utilizing the assembled colloidal structures for different applications (4, 5, 7, 8), considerably less effort is directed to understand the self-assembly mechanism itself in this process (17, 24). In particular, despite using the term “colloidal crystals” to highlight the microstructures’ long-range order, an analogy to atomic crystals, little is known regarding the crystallographic evolution of colloidal crystals in relation to the self-assembly process (3, 22, 25). The underlying mechanisms for the puzzling—yet commonly observed—phenomenon of the preferred growth along the close-packed <110> direction in evaporation-induced colloidal crystals are currently not understood (3, 25–29). The <110> growth direction has been observed in a number of processes with a variety of particle chemistries, evaporation rates, and matrix materials (3, 25–28, 30), hinting at a universal underlying mechanism. This behavior is particularly intriguing as the colloidal particles are expected to close-pack parallel to the meniscus, which should lead to the growth along the <112> direction and perpendicular to the <110> direction (16, 26, 31)^*.Preferred growth along specific crystallographic orientations, also known as texture development, is commonly observed in crystalline atomic solids in synthetic systems, biominerals, and geological crystals. While current knowledge recognizes mechanisms such as the oriented nucleation that defines the future crystallographic orientation of the growing crystals and competitive growth in atomic crystals (32–34), the underlying principles for texture development in colloidal crystals remain elusive. Previous hypotheses based on orientation-dependent growth speed and solvent flow resistance are inadequate to provide a universal explanation for different evaporation-induced colloidal self-assembly processes (3, 25–29). A better understanding of the crystallographically preferred growth in colloidal self-assembly processes may shed new light on the crystal growth in atomic, ionic, and molecular systems (35–37). Moreover, mechanistic understanding of the self-assembly processes will allow more precise control of the lattice types, crystallography, and defects to improve the performance and functionality of colloidal assembly structures (38–40).     

The unstructured linker of Mlh1 contains a motif required for endonuclease function which is mutated in cancers

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391–415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

DNA mismatch repair (MMR) acts on mispairs arising from DNA-replication errors, formation of homologous recombination intermediates, and some chemically modified DNA bases (1–3). During MMR, mispair recognition by MutS homologs, primarily Msh2-Msh6 and Msh2-Msh3 in eukaryotes (4–8), is required to recruit MutL homologs to mispaired DNA, primarily Mlh1-Pms1 in eukaryotes (called MLH1-PMS2 in humans) (1–3, 9). In organisms other than Escherichia coli and related bacteria (10), the MutL homologs have an endonuclease activity that specifically nicks double-stranded DNA on strands containing pre-existing nicks (11–13). Nicking by Mlh1-Pms1 in vitro is required for Exo1-mediated repair on substrates with a nick 3′ to the mispair, as formation of a strand-specific nick 5′ to the mispair allows the 5′–3′ exonuclease activity of Exo1 to excise the mispair (11–14). The absolute requirement of this Mlh1-Pms1 nicking activity in vivo is not well understood, as both 5′ and 3′ nicks relative to mispairs are likely already present on newly synthesized DNA strands (15, 16). One proposal suggests that Mlh1-Pms1 activity maintains single-stranded discontinuities, which appear to identify the newly synthesized strand, even in the presence of the competing activities, like DNA ligation and gap filling by DNA polymerases (15, 17).MutL homologs are comprised of an N-terminal GHKL family adenosine triphosphatase (ATPase) domain, a carboxyl-terminal dimerization domain, and a predicted unstructured linker domain that connects the folded N- and carboxyl-terminal domains (18–21). In Saccharomyces cerevisiae, the unstructured linkers of Mlh1 and Pms1 are ∼150 and 250 amino acids long, respectively (22). These linkers have a biased sequence composition with reduced hydrophobic amino acids, like the large (>50 amino acid) intrinsically disordered regions (IDRs) present in many proteins (23–25). IDRs often mediate intermolecular interactions, play functional roles, and sometimes become ordered when bound to partners (23–25).MutL homologs, including Mlh1-Pms1, form DNA-bound rings called sliding clamps following loading by MutS homologs, ATP binding, and dimerization of the N-terminal ATPase domains; these rings rapidly diffuse along the DNA axis (26–30). The extended length of the unstructured interdomain linkers has been suggested to allow these MutL homolog clamps to migrate past protein–DNA complexes, which are normally a barrier to MutS homolog clamps, although Msh2-Msh3 clamps appear to be able to open and close on encountering a protein–DNA complex and hop over it (26–29, 31, 32). Remarkably, cleavage of the S. cerevisiae Mlh1 linker in vivo causes increased mutation rates, suggesting that intact sliding clamps are important for MMR (22). The importance of the combined lengths of the Mlh1 and Pms1 linkers in vivo is suggested by the synergistic increases in mutation rate that have been observed when combining S. cerevisiae mlh1 and pms1 mutations that shorten the linkers (26). In contrast, some linker missense mutations, which do not alter linker lengths, cause MMR defects (22, 33–35). Moreover, deletions within the S. cerevisiae Mlh1 linker tend to cause MMR defects, whereas deletions in the S. cerevisiae Pms1 linker tend not to, except for the pms1-Δ390–610 deletion that eliminates almost the entire Pms1 linker, resulting in a mutant complex that cannot be recruited by Msh2–Msh6 to mispair-containing DNA and fails to bind to DNA under low ionic strength conditions (22). Together, the data suggest that length is only one requirement for the Mlh1 and Pms1 linkers and that the Mlh1 and Pms1 linkers differ in importance for MMR.Here, we have identified a motif in the Mlh1 linker, which spans residues 391–415, that is conserved from S. cerevisiae to humans and is required for MMR. Mutation of two of the residues in this motif, R401 and I409, to alanine caused an MMR defect, as did short deletions affecting other partially conserved residues within the motif. We found that the motif was functional when moved to different positions on the Mlh1 linker and when the distances between motif and the folded N- and carboxyl-terminal domains were altered. Moreover, moving a copy of the motif to the Pms1 subunit complemented the MMR defect caused by loss of the motif in Mlh1; in addition, swapping the Mlh1 linker with the Pms1 linker supported MMR. Mutant Mlh1-Pms1 complexes with amino acid substitutions in the conserved Mlh1 motif could not support reconstituted MMR reactions in vitro and were defective for Mlh1-Pms1 endonuclease activity but were recruited to mispair-containing DNA by Msh2-Msh6. Peptides encoding the conserved motif, but not control peptides, inhibited wild-type Mlh1-Pms1 endonuclease activity. Consistent with these observations, increased levels of Pms1-4GFP foci, which are MMR intermediates (36), were caused by mutations disrupting the conserved Mlh1 motif, similar to other mutations that reduce Mlh1-Pms1 endonuclease activity (36–38). Mutations of the motif were observed in human cancers, and these mutations disrupted MMR in vivo when modeled in the S. cerevisiae MLH1 gene. Taken together, these data are consistent with a requirement of the Mlh1 linker motif for Mlh1-Pms1 endonuclease activity in MMR, which could be due to an interaction of the motif with the DNA substrate, with the endonuclease active site, and/or with the endonuclease-activating PCNA.