Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages

When nucleotide-binding oligomerization domain–like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN^+/− mice were more responsive to inflammasome activation than those from GLMN^+/+ mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN.Inflammasome activation is a key defense mechanism against bacterial infection that induces innate immune responses such as caspase-1 activation and inflammatory cell death (1–3). Although the mechanisms through which various bacterial activities promote infection remain incompletely understood, some bacterial pathogens stimulate inflammasome activity by delivering cytotoxins, type III secretion (T3SS)-mediated effectors, T3SS components, flagellin, or cytotoxins to the host cell membrane and cytoplasm. These foreign components modify the host cell-surface architecture, induce membrane damage, subvert cell signaling, reorganize the actin cytoskeleton, and alter cell physiology (4) through interactions with various cytoplasmic receptors, e.g., nucleotide-binding oligomerization domain–like receptors (NLRs)—including NLRP1, NLR family CARD domain-containing 4 (NLRC4), NLR family pyrin domain-containing 3 (NLRP3), AIM2, IFI16, and RIG-1—as pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) (2, 3, 5). Upon recognition of these PAMPs and DAMPs, NLRs induce the assembly of inflammasomes, which are composed of NLR, apoptosis-associated speck-like protein (ASC), and inflammatory caspases such as caspase-1. Inflammasome assembly ultimately results in the extracellular release of IL-1β and IL-18 and induces inflammatory cell death (called “pyroptosis”) (6). For example, NLRP3 senses membrane rupture that occurs during infection with Listeria monocytogenes, Shigella, Salmonella typhimurium, Staphylococcus aureus, Neisseria gonorrhoeae, and Chlamydia spp. and upon exposure to bacterial pore-forming toxins, leading to caspase-1 activation (7–10). NLRC4 detects L. monocytogenes and S. typhimurium infection and stimulates caspase-1 activation (11–14). NLRC4 also senses flagellin and the T3SS rod components of Legionella pneumophila, Pseudomonas aeruginosa, Shigella, and S. typhimurium (11, 15–20) and the T3SS needle components of Chromobacterium violaceum, S. typhimurium, enterohemorrhagic Escherichia coli, Burkholderia thailandensis, and Shigella (21). Therefore, NLR inflammasomes act as major cytoplasmic pattern-recognition receptors and as central platforms that transmit alarm signals to a variety of downstream innate immune systems.Some bacterial pathogens, such as S. typhimurium (22) and Yersinia pseudotuberculosis (23–25), can induce macrophage death after they have fully replicated, promoting the egress of bacteria from their replicative compartments and the subsequent dissemination of bacteria into new host cells. This causal relationship suggests that these pathogens may benefit from and exert control over host cell death and the inflammatory response. In the case of Shigella, the bacteria rapidly induce macrophage cell death at early stages of infection, which is accompanied by NLR inflammasome activation and inflammatory cell death through a T3SS-dependent mechanism (19, 22). Previous studies indicated that during replication in macrophages, LPS, peptidoglycan, and T3SS rod or needle components of Shigella are recognized by the NLRC4 and NLRP3 inflammasomes (8, 19–21). Interestingly, the mode through which NLRs recognize Shigella infections seems to vary across different infection conditions. At a low infectious dose [e.g., a multiplicity of infection (MOI) of 10–25], bacteria induce rapid NLRC4–caspase-1–dependent pyroptosis at 2–3 h postinfection through the recognition of the T3SS components or some uncharacterized T3SS-delivered substance(s) (19, 22). However, at a high infectious dose (e.g., an MOI over 50) and at later time points (6 h postinfection), the bacteria induce NLRP3-dependent but caspase-1–independent necrosis-like cell death with inflammation (called “pyronecrosis”) (8). Because pyroptosis results in the release of intracellular contents, including proinflammatory cytokines and chemokines, and because, in the case of Shigella, macrophage death is a prerequisite for the subsequent infection of surrounding epithelial cells (19, 26), it remains unclear whether Shigella-mediated rapid cell death is beneficial to the pathogen or to the host. Nevertheless, these studies strongly suggest that the bacteria deploy multiple mechanisms to manipulate macrophage cell-death pathways in a T3SS-dependent manner.Shigella flexneri, e.g., the YSH6000 strain, possesses three invasion plasmid antigen H (ipaH) genes, ipaH9.8, ipaH7.8, and ipaH4.5, on a large virulence plasmid (27, 28). These IpaH proteins, which originally were identified in the S. flexneri M90T strain (29, 30), recently were found to act as enzyme 3 (E3) ubiquitin ligases (31) and were thus named “novel E3 ligases” (32). The ipaH cognate genes are distributed among various Gram-negative bacterial pathogens, including Shigella, Salmonella, Yersinia, Edwardsiella ictaluri, Bradyrhizobium japonica, Rhizobium sp. strain NGR234, Pseudomonas putida, Pseudomonas entomophila, Pseudomonas fluorescens, and Pseudomonas syringae (31). IpaH protein family members share structural and functional similarity; they are composed of an N-terminal leucine-rich repeat (LRR) and a highly conserved C-terminal region (CTR) (33, 34). The conserved CTR contains a Cys residue, which is critical for E3 ubiquitin ligase activity (31, 35, 36). Each of the IpaH family effectors characterized to date (e.g., Shigella IpaH9.8 and IpaH2077, Salmonella SlrP, SspH1, and SspH2, Yersinia YopM, and Rhizobium Y4fR and BIpM) has distinct host protein targets in different host cell types. Some act as effectors to attenuate host inflammatory responses, whereas others modulate host defense responses in plants (37, 38). The existence of multiple effectors with E3 ligase activity suggests that an array of E3 ligases is required to promote bacterial infection and antagonize host innate defense responses.Fernandez-Prada et al. (39) previously reported that Shigella lacking the ipaH7.8 gene are less capable than the WT strain of escaping the phagocytic vacuole of macrophages and that Shigella infection of macrophages induces apoptotic-like cell death. Paetzold et al. (40) subsequently showed that Shigella lacking the ipaH7.8 gene had no effect on phagosome escape compared with the WT strain, but bacterial-induced cytotoxicity was low compared with that of the WT strain. Although the biological significance of IpaH7.8 as an E3 ubiquitin ligase during Shigella infection remains to be elucidated, these studies suggested that IpaH7.8 is involved in inducing macrophage cell death.In this context we wished to clarify the pathological role of IpaH7.8 as an E3 ubiquitin ligase in Shigella infection of macrophages and the modality of cell death. Here we provide evidence that IpaH7.8 potentiates macrophage killing in an IpaH7.8 E3 ligase-dependent manner, in which E3 ligase activity triggers NLR inflammasome-mediated macrophage cell death by targeting glomulin/FAP68 (GLMN); this activity ultimately appears to benefit the pathogen.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.Infectious diseases typically arise when pathogens grow to high tissue loads, causing extensive damage and immunopathology. An outstanding example is Shigella flexneri, which rapidly grow from a small infectious dose of 10–100 bacteria (1) to intestinal loads causing life-threatening bloody diarrhea (bacillary dysentery) within a few hours (2, 3). This vigorous Shigella growth occurs inside human colon epithelial cells and requires an integrated Shigella pathogenesis program, including a type three secretion system encoded on the Shigella virulence plasmid. Using this system, Shigella translocates enzymes into the host cell cytosol, where they target key cellular functions, allowing Shigella to enter the host cell and escape bacterial killing by innate immune responses (4). After Shigella reaches the host cell cytosol, many virulence factors are down-regulated (5), and Shigella starts rapid proliferation.Biomass generation at such high rates depends on extensive exploitation of intracellular host nutrients (6). The host cell cytoplasm contains hundreds of metabolites, but it is unclear which of these potential nutrients Shigella uses, how the host cell can supply them at sufficiently high rates to support rapid Shigella growth, and why host cells can sustain viability while being vigorously exploited by intracellular Shigella. For related enteroinvasive Escherichia coli, previous research has shown that glucose and other host metabolites, such as diverse amino acids, can be incorporated into the biomass of these closely related pathogens (7). However, quantitative data are still lacking, and energy production, which is usually a major part of nutrient use (8), could not be analyzed because of technical limitations.In general, pathogen metabolism has been recognized as a fundamentally important aspect of infectious diseases, but available data are mostly restricted to qualitative presence/absence of enzymes in pathogen genomes and metabolite or gene expression profiles in various infection models (9–13). Comprehensive quantitative studies on pathogen nutrition, metabolism, and growth are largely lacking. This limited knowledge reflects, in part, the fact that suitable methodologies are just becoming available. In this study, we combined various metabolomics approaches, proteomics, and microbial genetics to elucidate the metabolic basis of Shigella rapid growth in infected human host cells.     

Antibody WN1 222-5 mimics Toll-like receptor 4 binding in the recognition of LPS

Escherichia coli infections, a leading cause of septic shock, remain a major threat to human health because of the fatal action to endotoxin (LPS). Therapeutic attempts to neutralize endotoxin currently focus on inhibiting the interaction of the toxic component lipid A with myeloid differentiating factor 2, which forms a trimeric complex together with Toll-like receptor 4 to induce immune cell activation. The 1.73-Å resolution structure of the unique endotoxin-neutralizing protective antibody WN1 222-5 in complex with the core region shows that it recognizes LPS of all E. coli serovars in a manner similar to Toll-like receptor 4, revealing that protection can be achieved by targeting the inner core of LPS and that recognition of lipid A is not required. Such interference with Toll-like receptor complex formation opens new paths for antibody sepsis therapy independent of lipid A antagonists.LPS from Gram-negative bacteria is the major etiological agent of septic shock, which is a serious and often fatal dysregulation of the innate immune response that affects 750,000 people in the United States annually (1). Infection with Escherichia coli, together with Klebsiella, Neisseria, and Pseudomonas, are the most frequent isolates in septic shock (2). A key event initiating the shock cascade is the induction of the innate immune response by the complex formation of a symmetric “m”-shaped multimer composed of two copies of Toll-like receptor 4 (TLR4), myeloid differentiating factor 2 (MD-2), and LPS (3, 4). In a landmark publication, the structure of TLR4-MD-2 bound to LPS (3) was recently described.LPS is composed of an acylated glucosamine phosphate disaccharide (i.e., lipid A), which is the endotoxic principle of LPS, a core oligosaccharide (core-OS) and a distal O-polysaccharide (O-PS) often composed of repeating units (Fig. 1A). Whereas the O-PS is structurally heterogeneous, with more than 180 reported E. coli serotypes (5), the core region is composed of a more conserved structure commonly divided into the inner Kdo-heptose and outer hexose regions (6).Open in a separate windowFig. 1.Structures of LPS and the shape of the combining site. (A) Structure of E. coli R2 dodecasaccharide-P₄, representing the core and lipid A of the LPS from Enterobacteria commonly associated with septic shock. (B) Stereo views of electron density corresponding to 10 sugar residues of the core antigen (the lipid A moiety is disordered) contoured at 1.0 σ.Recognition of LPS leads to a paramount immunological defense reaction caused by the activation of a complex network of immunological mediators. Attempts to control the clinical development of sepsis by neutralizing the most important proinflammatory mediators have failed, including the recent withdrawal of recombinant activated protein C (Xigris). A promising antagonistic lipid candidate called Eritoran (E5564; Eisai) (7) also recently failed in clinical trials, and alternative treatments are urgently needed. The discovery of TLR4 as the principal receptor for endotoxins (8) has stimulated the development of drugs aiming at its down-regulation (9) through interference of LPS–TLR4–MD-2 complex formation (4, 10–12).Antisera specific for O-PS have been shown to protect against LPS lethality (13); however, the diversity of enterobacterial O-PS together with the rapid onset of septic shock have hindered their introduction into clinical practice (11).The hypothesis that mAbs specific to the conserved inner core region or lipid A would be protective against a wide range of serovars and even different species was put forward (14) after the discovery of structural similarities within their respective LPSs. WN1 222-5 is the only neutralizing antibody reported to date that displays specificity for an epitope within the structurally conserved region of LPS from a large number of pathogenic E. coli, Salmonella, Shigella, and Citrobacter serovars (15). Further, WN1 222-5 has been shown to inhibit the recognition and uptake of LPS by cells expressing coreceptor mCD14, likely by hindering the transfer of LPS to TLR4–MD-2 (16).WN1 222-5 has been shown to inhibit the inflammatory cascade in in vivo studies of septic shock, in which it prevents the pyrogenic response in rabbits, inhibits the Limulus amoebocyte lysate assay, and inhibits LPS-induced monokine secretion (15–17).The difficulties in growing crystals of antibodies in complex with carbohydrate antigens has led to relatively few reported structures (18–21), leading, for example, to increased use of structure prediction tools such as molecular dynamics modeling (22). Thus, in contrast to their great immunological significance during infectious disease, still relatively little is known about carbohydrate recognition by antibodies at the structural level. Whereas cavity- or groove-shaped antibody-combining sites have been observed in most cases, a unique mechanism of binding has been observed for the HIV-1 neutralizing antibody 2G12, binding clusters of carbohydrates from the silent face of gp120 by using “domain swapping” (19, 23, 24).The structural analysis of antibodies Se155-4 and S20-4 against O-PS of Salmonella enterica and Vibrio cholerae, respectively, have revealed structural insights into the high specificity for a particular serotype (20, 25). However, because of their specificity, antibodies against O-PS are of limited use for the treatment of infectious disease. Nevertheless, structures of antibodies in complex with large carbohydrate antigens have revealed critical insights for vaccine development. The protective antibody F22-4 in complex with an 11-sugar segment from the O-PS of Shigella flexneri serotype 2a (26) allowed the design of new immunogens.Most attempts in obtaining antibodies that are broadly reactive with a wide variety of LPSs from different Gram-negative bacteria have failed, and epitopes within the deeper core region of LPS have been regarded as not accessible to antibodies in WT LPSs of infectious bacteria. To provide detailed insight on a unique cross-reactive and neutralizing ability, the Fab from WN1 222-5 in complex with a complete core-OS of LPS from E. coli has been crystallized and its structure determined to 1.73-Å resolution.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein–glycan or protein–protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host–glycan:bacterial–glycan pairs with equilibrium dissociation constants (K_D) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems.Host surface glycosylation is ubiquitous and is targeted by pathogenic bacteria, viruses, fungi and parasites for adherence and toxin binding and by glycosidases (1). Escherichia coli type 1 fimbriae, FimH, is one of the most widely studied glycan-recognizing protein adhesins, with specificity for monomannose to oligomannose structures with the variability of the mannose structure bound leading to different tissue tropism (2). Other glycan-recognizing adhesins expressed by bacteria include the following: Pseudomonas aeruginosa lectins 1 and 2 (PA-IL and PA-IIL) that have specificity for galactose and fucose, respectively (3); Helicobacter pylori SabA, specific for sialic acid containing glycoconjugates including sialyLewis X; and BabA-specific for fucosylated glycoconjugates including Lewis B (4, 5). Although there are numerous known glycan binding adhesins, the adhesins of some bacteria that interact with host surface glycans remain unknown.Direct interactions between surface glycans (glycan:glycan interactions) have been reported in sea sponges as heterogenous glycan interactions, and in mouse embryo development and cancer where homodimers of Lewis X (LeX) or ganglioside structures play a role in cell adhesion and growth factor receptor interactions (6, 7). Outside of these reports, glycan:glycan interactions, when noted, have generally been considered to be low-affinity, weak interactions (8) that precede high-affinity protein:glycan or protein:protein interactions (1, 2, 5, 9).Interestingly, there are specific reports of several bacteria expressing truncated surface polysaccharides and oligosaccharides that are significantly less adherent than wild-type equivalents (10, 11), or that their adherence can be blocked by extracted LOS/LPS (10), indicating a role for bacterial surface glycans in adherence to host cells. This decreased adherence of rough strains or blocking of adherence using the free lipooligosaccharide (LOS)/lipopolysaccharide (LPS) in both cell-based and animal infection models has been noted in a range of Gram-negative bacteria including Campylobacter jejuni, Haemophilus influenzae, Salmonella typhi, Salmonella enterica serovar Typhimurium, E. coli, Shigella flexneri, Pseudomonas aeruginosa, and Serratia marcescens (10, 12–20). Blocking of surface glycans with antibodies has also been shown to inhibit adherence and invasion of cell layers in a range of bacteria, including S. flexneri (21–23). The cellular receptors for adherence via these bacterial surface glycans have not been identified. To address the hypothesis that there may be direct interactions between bacterial and host glycans that mediate adherence, we conducted glycan microarray screening of four different species of pathogenic bacteria with well-characterized surface glycan structures: C. jejuni, H. influenzae, S. typhimurium, and S. flexneri. These studies included whole live bacteria expressing wild-type and LOS/LPS truncation mutants, as well as purified LOS/LPS from the same set of bacteria.     

TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ

Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia–neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K⁺ concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30–100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.Microglia are tissue-resident macrophages in the CNS that become activated in most brain disorders, such as bacterial meningoencephalitis, multiple sclerosis, and Alzheimer’s disease (1, 2). Activation of microglia features changes in morphology and receptor expression, antigen presentation, cytokine release, migration, and phagocytosis, and it ranges from proinflammatory and potentially neurotoxic to anti-inflammatory and neuroprotective phenotypes (1, 3, 4). The mechanisms that control the transition of microglia to reactive phenotypes, including the impact on neuronal function, are mostly unknown, however (5–7).Sensing of microbial or modified endogenous ligands by microglia is mediated by innate pattern recognition receptors, such as scavenger receptors and Toll-like receptors (TLRs). A prime example is TLR4, which acts with CD14, MD-2, and lipopolysaccharide (LPS)-binding protein in recognizing LPS, a cell wall component of Gram-negative bacteria (8, 9). TLR4 is also central to microglial recognition of amyloid-β peptide, which is thought to be part of the inflammatory response in Alzheimer’s disease (7, 10).LPS has been widely used to study the molecular mechanisms of microglial activation in inflammatory neurodegeneration (1–3). In primary monocultures and microglia-neuron cultures, LPS exposure alone or in combination with IFN-γ for a “booster” triggers the massive release of proinflammatory and cytotoxic factors, such as TNF-α, IL-6, and nitric oxide (NO), finally resulting in neuronal death (8, 11–18). Similar effects were observed in vivo after intracerebral administration of LPS (19–21). These and other studies have contributed to the concept that microglial TLR4 activation with LPS (i.e., with a single pathogenic stimulus) is sufficient to induce neurodegeneration (22, 23); however, this concept is biologically risky, and has been questioned in some experimental works and reviews (2–4, 11, 24, 25).Most previous studies focused on two aspects of microglial TLR4 activation with LPS: (i) the properties of the reactive microglial phenotype(s) and (ii) the degree of neurodegeneration. For this purpose, either simple culture systems or in vivo models, in which interactions with leukocytes infiltrating from the blood are inevitable, have been used (1, 4). Thus, it is widely unknown how TLR4 and IFN-γ receptor signaling in microglia individually contribute to neurotoxicity and neurodegeneration in situ. This aspect is highly relevant for several neurologic and psychiatric disorders. Moreover, concomitant alterations in neuronal information processing (i.e., dysfunction in excitatory pyramidal cells and inhibitory GABAergic interneurons, including astrocytes) have been little explored (25–27).We rigorously addressed these fundamental questions in postnatal neuronal tissue (1, 4). To mimic microglial confrontation with LPS in situ and, notably, in the absence of infiltrating leukocytes, we used organotypic hippocampal slice cultures that feature highly preserved cytoarchitectures and complex neuronal network functions (5, 28). Microglial interaction with infiltrating T helper type 1 (Th1) cells and/or natural killer (NK) cells was mimicked by recombinant IFN-γ administration.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.