Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in the United States (1, 2). Over the past decade, morbidity and lethality from CDI have increased (3, 4), and the need for new treatment options has become a priority.The pathology associated with CDI is associated with the activities of two large, glucosylating toxins, TcdA and TcdB (5). Upon binding to the colonic epithelium, these toxins induce the fluid secretion, immune cell influx, and tissue damage associated with clinical manifestations of CDI (5). TcdA and TcdB have four functional domains: an N-terminal glucosyltransferase domain (GTD), an autoprotease domain, a pore-forming and delivery domain, and a combined repetitive oligopeptides (CROPS) domain, which extends from around residue 1830 to the C terminus and has been implicated in receptor binding. The toxins enter cells by receptor-mediated endocytosis (6). Acidification of the endosome is thought to trigger a structural change in the delivery domain, allowing for pore formation and translocation of the GTD into the cytosol (7, 8). Activation of the autoprocessing domain by eukaryotic inositol hexakisphosphate results in the release of the GTD into the cell, allowing access to substrates (8). The GTD transfers a glucose from UDP glucose onto the switch I region of Rho family GTPases such as Rho, Rac1, and Cdc42 (9, 10). These modifications cause a cytopathic effect resulting from rearrangement of the actin cytoskeleton and can lead to apoptosis (11). At higher concentrations, TcdB is also capable of inducing the production of reactive oxygen species, resulting in cell death by a necrotic mechanism (12, 13). We speculate that both mechanisms are important in the context of disease; the cytopathic effects promote inflammation and disruption of the tight junctions, whereas the TcdB-induced necrosis contributes to the colonic tissue damage observed in severe cases of CDI.Although TcdA and TcdB are homologs, they appear to perform separate, nonredundant functions (14, 15). TcdA and TcdB are thought to have different receptors, based on sensitivity differences among cell types in vitro (16–19). Multiple receptors for TcdA have been proposed including Gal alpha 1–3Gal beta 1–4GlcNAc, blood antigens I, X, and Y, rabbit sucrase isomaltase, and gp96 (18, 20–22). The TcdA CROPS domain is thought to play a role in binding cell surface carbohydrates (18, 23, 24). Antibodies against the CROPS domains of TcdA and TcdB can block intoxication (25, 26), and excess TcdA CROPS domain can compete with TcdA holotoxin for cell binding (27). At the same time, truncations of TcdA and TcdB that lack the CROPS domains are still capable of intoxicating cells (7, 28, 29) and a homologous toxin from Clostridium perfringens, TpeL, lacks a CROPS domain entirely (29, 30). A receptor for TpeL has been identified (29), suggesting that a receptor-binding site for other large clostridial toxins could exist outside of the CROPS. A recent report indicates that chondroitin sulfate proteoglycan 4 (CSPG4) mediates TcdB-induced cytopathic and apoptotic events in HeLa and HT29 cells (31). CSPG4 does not mediate the necrotic effects that occur at higher TcdB concentrations and CSPG4 binds TcdB outside the CROPS; these observations are consistent with a dual receptor hypothesis. This study represents an independent effort to define the cellular factor(s) responsible for TcdB binding and toxicity.     

Structure of a bacterial toxin-activating acyltransferase

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host–cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.Pathogenic bacteria secrete pore-forming protein toxins (PFTs) that target tissue and immune cell membranes to aid colonization and survival during infections, subvert cell signaling, induce apoptosis, and promote cell lysis (1–8). Among Gram-negative bacteria, large PFTs are secreted by pathogenic species of Pasteurella, Actinobacillus, Proteus, Morganella, Moraxella, and Bordetella, exemplified by the 110-kDa hemolysin (HlyA) of uropathogenic and enterohemorrhagic Escherichia coli. These toxins play important roles in cystitis and pyelonephritis, hemorrhagic intestinal disease, periodontitis, pneumonia, septicemia, whooping cough, and wound infections (4), and unusually they are made as an inactive protoxin, requiring posttranslational activation before export (9–12).Reconstituting the toxin activation reaction in vitro some time ago demonstrated that the essential modification is a novel fatty acid acylation, affected by a specialized coexpressed toxin-activating acyltransferase, in E. coli HlyC, that uses acyl-acyl carrier protein (acyl-ACP) as the fatty acid donor (4, 13, 14). The acyltransferase does not share significant sequence identity with other bacterial and eukaryotic enzymes, and cellular acyltransferases from either the host or pathogen cannot substitute for HlyC in toxin activation. HlyC independently binds two separate 50- to 80-aa transferase recognition domains (15), each encompassing one of the internal target lysines K564 and K690 of E. coli protoxin HlyA, which are acylated by amide linkage, heterogeneously with fatty acids containing 14, 15, and 17 carbon chains (16, 17). Loss of the HlyC binding domain or substitution of protoxin K564 and K690 prevents fatty acyl modification and abrogates all toxin activity (14) as does loss of the transferase (18).Acylation is essential to the entire family of pore-forming toxins, Bordetella pertussis proCyaA lysine acylation has also been demonstrated (19), and the toxin-activating acyltransferases (which we now call TAATs) have high sequence similarity and cross-activate other protoxins (4, 20–22). The TAAT activation mechanism is seemingly unique, and extensive site-directed mutagenesis has so far only identified a single potentially catalytic residue, His23 of HlyC (23–25). Structural information is essential to understand the toxin activation mechanism and assess TAATs as a potential target for developing antivirulence compounds that do not affect the host commensal flora. Here, we determine the TAAT crystal structure, solution state, and likely active site.     

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study’s in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.Transport of protein across the cell membrane is a complex process that usually involves multipart translocation machineries. Many protein toxins from poisonous plants or from pathogenic bacteria are able to penetrate into the cytosol of their target cells where they exert their toxic effects. Some of these toxins exploit the endogenous cellular machinery of endocytosis and intracellular sorting to gain access to the cell cytosol, but others carry their own translocation apparatus (1–4). These latter toxins provide a unique opportunity to analyze the molecular mechanisms and the physicochemical principles underlying polypeptide transport across biological membranes. Studies combining structural, biochemical, and electrophysiological approaches have begun to unravel the various strategies developed by these toxins to deliver their catalytic moieties across the cell membranes (5–10).The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the few known toxins able to invade eukaryotic cells through a mechanism of direct translocation across the plasma membrane of the target cells (11–13). CyaA is an essential virulence factor of B. pertussis that is secreted by virulent bacteria and able to enter into eukaryotic cells, where, on activation by endogenous calmodulin (CaM), it catalyzes high-level synthesis of cAMP, which in turn alters cellular physiology (14–16). CyaA is a 1,706-residue-long bifunctional protein organized in a modular fashion (Fig. 1A); the ATP-cyclizing, CaM-activated catalytic domain (AC) is located in the 400 amino-proximal residues, whereas the carboxyl-terminal 1,306 residues are responsible for the hemolytic phenotype of B. pertussis (17–20).Open in a separate windowFig. 1.Principle of CyaA translocation assay on tBLM/CaM assembly. (A) Scheme of CyaA toxin structure showing the three major domains: the catalytic domain, AC; the hydrophobic region, H, responsible for insertion of CyaA into the membrane; and the Ca²⁺-binding, RTX-containing domain, RD. (B) Schematic illustration of the approach used to monitor CyaA translocation across the tBLM. (C) Schematic representation of the SPR sample cell cross-section and tBLM/CaM construction.The C-terminal “hemolysin” moiety contains, between residues 500 and 750, several hydrophobic segments that are predicted to adopt alpha-helical structures and to insert into membranes to create the cation-selective pores responsible for the hemolytic activity (20, 21). The C-terminal part of the molecule (RD; residues 1,000–1,706) is involved in toxin binding to a specific cellular receptor (CD11b/CD18) (22, 23). This domain consists of approximately 40 copies of a calcium-binding, glycine- and aspartate-rich nonapeptide repeat (residues 1,014–1,613) characteristic of a large family of bacterial cytolysins known as repeat-in-toxin (RTX) toxins (11, 13, 24, 25).The CyaA toxin is synthesized as an inactive precursor, proCyaA, which is converted into the active toxin form (CyaA) on specific acylation of two lysine residues (26, 27). Then CyaA is secreted across the bacterial envelope by a dedicated type I secretion machinery and binds to the CD11b/CD18 integrin expressed by a subset of leukocytes including neutrophils, macrophages, and dendritic cells (22, 28–30). However, CyaA can also invade a wide variety of cells that do not express this receptor, albeit with a lower efficiency (19, 31–35).The most unique property of CyaA is its capability to deliver its N-terminal catalytic domain directly across the plasma membrane of the eukaryotic target cells, a process that occurs independently of the CD11b/CD18 receptor (11–13). It is believed that CyaA first inserts its hydrophobic segments into the plasma membrane and then delivers its catalytic domain across the plasma membrane into the cell cytosol (19, 31, 32) (Fig. 1B). Previous studies have shown that the translocation process is dependent on the temperature (occurring only above 15 °C), the membrane potential of the target cells, and the presence of calcium ions in the mM range (32, 36). Inside the cell, on binding to CaM with a subnanomolar affinity, CyaA is stimulated by more than 1,000-fold and exhibits a high catalytic rate (k_cat > 2,000 s⁻¹) to produce supraphysiologic levels of cAMP (12, 19, 37).How the hydrophilic CyaA catalytic domain of approximately 400 residues is able to pass across the hydrophobic barrier of the plasma membrane remains largely unknown, and whether specific eukaryotic proteins and/or cell membrane components are involved in this process is also unclear (19, 32, 35, 38, 39). To characterize the molecular mechanisms of CyaA translocation across the membrane, we performed a functional in vitro assay that exploits a recently designed biomimetic membrane assembly composed of a bilayer membrane (tBLM) tethered over an amino-grafted gold surface derivatized with CaM (40). This multilayer biomimetic assembly exhibits the fundamental feature of an authentic biological membrane in creating a continuous, yet fluid phospholipidic barrier between two distinct compartments: a cis side, corresponding to the extracellular milieu, and a trans side, marked by the cytosolic protein CaM (Fig. 1C). We monitored the binding of CyaA to the tBLM by surface plasmon resonance (SPR) spectroscopy, and detected the translocation of the catalytic domain across the bilayer by CyaA activation by the immobilized CaM. With this highly sensitive assay, translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and application of a negative transmembrane potential, in agreement with previous studies on eukaryotic cells (36).Our results demonstrate that CyaA does not require any specific eukaryotic components apart from CaM to translocate across a membrane. They also suggest that the catalytic domain may be electrophoretically transported across the bilayer in a calcium-dependent manner. This study provides a direct in vitro demonstration of a toxin translocation across a tBLM (41) and suggests that the biomimetic tBLM/CaM structure may be a useful tool for characterizing the molecular mechanisms of protein translocation across biological membranes under precisely defined conditions.     

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.The success of a broad array of microbial pathogens depends on their ability to gain entry into and/or transport proteins into the cytosol of host cells. Intracellular-acting bacterial toxins have evolved to take advantage of numerous host-mediated entry mechanisms (1), making these toxins ideal tools for studying endocytosis and vesicular trafficking. Indeed, the use of bacterial toxins has contributed to many key discoveries, including membrane recycling, clathrin-independent endocytosis, and retrograde transport (2). Compounds that inhibit entry of ricin, Shiga toxin, and Pseudomonas aeruginosa exotoxin A (ExoA) into host cells have been identified (3–5). These small molecules exhibit varied mechanisms of action, including blockade of retrograde toxin trafficking at the early endosome–trans Golgi network (TGN) junction, morphological disruption of the Golgi apparatus, and inhibition of the toxin active site. Small molecules that disrupt toxin binding, entry, trafficking, and host response can serve not only as probes to dissect such eukaryotic cellular pathways, but also are potential therapeutics for infectious and genetic diseases.Bacillus anthracis, the causative agent of the disease anthrax, secretes binary toxins that enter host cells and disrupt physiological processes. Lethal factor (LF) is a Zn²⁺-dependent metalloprotease that cleaves mitogen-activated protein kinase kinases (MAPKKs) 1–4, 6, and 7 (6, 7) and Nlrp1b (8–10) and reproduces many pathologies of anthrax when injected into laboratory animals (11, 12). The cellular entry of LF is dependent on a cell-binding and translocation subunit known as protective antigen (PA). PA is an 83-kDa protein that is cleaved by host proteases into 63- and 20-kDa fragments, allowing oligomerization of the toxin into a prepore (13). The PA oligomer can then bind up to four monomers of LF, forming a holotoxin complex (14, 15). Two cellular toxin receptors, TEM8 and CMG2, mediate toxin binding and endocytic uptake (16, 17). Acidification of the lumen of the late endosome drives a conformational change in the prepore, resulting in insertion into the endosomal membrane and translocation of LF into the cytosol (18–20). Alternatively, LF may be translocated to the interior of intraluminal vesicles and transported to the late endosome via multivesicular bodies in a process dependent on COPI and ALIX (21). The vesicular membranes then fuse with the limiting endosomal membrane and thereby deliver LF to the cytosol (21).Despite substantial effort to define binding and entry mechanisms used by lethal toxin (LT), much is still unknown about how it, and indeed many other toxins and viruses, gain access to the host cytosol. To address this, we performed a high-throughput screen to identify small molecules that block cellular entry of LT. Here, we report the identification and characterization of a compound that blocks trafficking of various toxins and viruses to acidified endosomes.     

Identification of divergent type VI secretion effectors using a conserved chaperone domain

The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.Protein secretion systems play a pivotal role in bacterial interspecies interaction and virulence (1, 2). Of the known secretion systems in Gram-negative bacteria, the type VI secretion system (T6SS) enables bacteria to compete with both eukaryotic and prokaryotic species through delivery of toxic effectors (2–4). The T6SS is a multicomponent nanomachine analogous to the contractile bacteriophage tail (5). First characterized in Vibrio cholerae (6) and Pseudomonas aeruginosa (7), the T6SS has now been identified in ∼25% of Gram-negative bacteria, including many important pathogens (2, 8), and has been implicated as a critical factor in niche competition (9–11).The T6SS structure is composed of an Hcp inner tube, a VipAB outer sheath that wraps around the Hcp tube, a tip complex consisting of VgrG and PAAR proteins, and a membrane-bound baseplate (2, 4, 12). Sheath contraction drives the inner Hcp tube and the tip proteins, VgrG and PAAR, outward into the environment and neighboring cells (13, 14). The contracted sheath is then dissembled by an ATPase ClpV and recycled for another T6SS assembly and contraction event (12, 15, 16). Two essential T6SS baseplate components, VasF and VasK, are homologous to the DotU and IcmF proteins of the type IV secretion system (T4SS) in Legionella pneumophila (17).Bacteria often possess multiple copies of VgrG and PAAR genes that form the tip of T6SS, and deletion of VgrG and PAAR genes abolishes T6SS secretion (14). Some VgrG and PAAR proteins carry functional extension domains and thus act as secreted T6SS effectors, as exemplified by the VgrG1 actin cross-linking domain (6), VgrG3 lysozyme domain in V. cholerae (18, 19), and the nuclease domain of the PAAR protein RhsA in Dickeya dadantii (20). Known T6SS effectors can target a number of essential cellular components, including the actin and membrane of eukaryotic cells (18, 21, 22) and the cell wall, membrane, and DNA of bacterial cells (3, 18–20, 23, 24). Each antibacterial effector coexists with an antagonistic immunity protein that confers protection during T6SS-mediated attacks between sister cells (3, 18, 24). Interestingly, T6SS-mediated lethal attacks induce the generation of reactive oxygen species in the prey cells (25), similar to cells treated with antibiotics (26, 27).For non-VgrG/PAAR–related effectors, their translocation requires either binding to the inner tube Hcp proteins as chaperones or binding to the tip VgrG proteins (2, 14, 28). T6SS-dependent effectors can be experimentally identified by comparing the secretomes of WT and T6SS mutants (3, 29–31) and by screening for T6SS-encoded immunity proteins (18). Because known effectors lack a common secretion signal, bioinformatic identification of T6SS effectors is challenging. A heuristic approach based on the physical properties of effectors has been used to identify a superfamily of peptidoglycan-degrading effectors in bacteria (32). A recent study identified a common N-terminal motif in a number of T6SS effectors (31). However, this motif does not exist in the T6SS effector TseL in V. cholerae (18).In this study, we report that VC1417, the gene upstream of tseL, encodes a protein with a highly conserved domain, DUF4123. We show that VC1417 is required for TseL delivery and interacts with VgrG1 (VC1416) and TseL. Because of the genetic linkage of VC1417 and TseL and its importance for TseL secretion, we postulated that genes encoding the conserved DUF4123 domain proteins are generally located upstream of genes encoding putative T6SS effectors. Using the conserved domain sequence, we bioinformatically predicted a large family of effector proteins with diverse functions in Gram-negative bacteria. We validated our prediction by the identification and characterization of a new secreted effector TseC and its antagonistic immunity protein TsiC in Aeromonas hydrophila SSU. Our results demonstrate a new effective approach to identify T6SS effectors with highly divergent sequences.     

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.Toxins were recognized more than a century ago to play a major role in bacterial infectious diseases (1). Subsequently, hundreds of toxins from pathogenic bacteria have been characterized. Based on bioinformatic analysis of its genome, toxins appear to be absent from Mycobacterium tuberculosis (2–4), the causative agent of tuberculosis, a devastating disease with nine million new cases every year (5). Survival within host macrophages is a key trait enabling M. tuberculosis to persist in the human body (6), where it can reactivate after decades of quiescence (7). This so-called latent infection is poorly understood and is one of the reasons why tuberculosis remains a global public health problem. Alveolar macrophages engulf inhaled M. tuberculosis, contribute to killing of the bacteria, reduce inflammation of lung tissue, and limit uptake of M. tuberculosis by migratory dendritic cells to prevent bacterial dissemination (8). However, M. tuberculosis has evolved effective strategies to subvert this bactericidal response (6, 9). Death of M. tuberculosis-infected macrophages is caused by two processes: necrosis and apoptosis. Necrosis is characterized by metabolic collapse and loss of membrane integrity and is used by M. tuberculosis to exit destroyed cells, evade host defenses, and disseminate to other tissues and eventually to new hosts (10). By contrast, apoptosis of the infected macrophages helps the host to control the bacterial infection (11). Virulent M. tuberculosis strains induce a necrosis-like cell death and concomitantly suppress apoptosis of macrophages (12). Although M. tuberculosis is known to secrete virulence factors that interfere with phagosome maturation (9), it is unknown how M. tuberculosis kills macrophages.Gram-negative bacterial pathogens use complex nanomachines such as type I–VI secretion systems to secrete effector proteins mediating host cell death and subverting immune responses (13). Similarly, proteins secreted by M. tuberculosis need to cross both an inner and an outer membrane (14), a barrier of notoriously low permeability in M. tuberculosis (15). However, the only known secretion systems capable of translocating proteins across both M. tuberculosis membranes are the type VII secretion systems encoded by esx operons. Although inner membrane proteins of ESX secretion systems have been characterized (16), channel proteins that are required for protein translocation across the outer membrane are currently unknown in M. tuberculosis. We hypothesized that deletion or inactivation of outer membrane channel proteins in M. tuberculosis may result in increased antibiotic resistance, as has been described for Gram-negative bacteria and Mycobacterium smegmatis (17, 18). Here we show that this approach identified a protein that enables uptake of small, hydrophilic molecules via its N-terminal pore domain and induces host cell necrosis by its secreted toxic C-terminal domain (CTD).     

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.     

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues—clustered between amino acids 1,035 and 1,107—that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming “hotspots” of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.The primary virulence determinants of pathogenic Clostridium difficile are two protein toxins, TcdA and TcdB, which are responsible for the symptoms associated with infection, including diarrhea and pseudomembranous colitis (1). TcdA and TcdB are large (i.e., 308 and 270 kDa, respectively) homologous toxins (sharing 48% sequence identity) that appear to intoxicate target cells using a strategy that is similar in principle to that described for a number of smaller A–B toxins, such as anthrax toxin (2) and diphtheria toxin (DT) (3). In addition to a cytotoxic enzymic A domain and receptor-binding B domain responsible for binding and translocating the A domain into cells, TcdA and TcdB are additionally equipped with an internal autoprocessing domain that proteolytically cleaves and releases the N-terminal glucosyltransferase domain in response to intracellular inositol hexakisphosphate (4).The series of events leading to the delivery of the A domain into cells begins with toxin binding to an as yet unidentified receptor on target cells via the C-terminal receptor-binding domain (i.e., the B domain), which triggers toxin internalization into acidified vesicles via clathrin-mediated endocytosis (5). In the endosome, cryptic regions from within the large ∼1,000-aa translocation domain emerge and insert into the endosomal membrane, creating a pore that is believed to enable translocation of the N-terminal glucosyltransferase (i.e., the A domain) into the cytosol. Processed and released A chains enzymatically glucosylate and thereby inactivate intracellular Rho and Ras family GTPases (6, 7) leading first to cytopathic effects (i.e., cell rounding) (8), and later, cytotoxic effects (i.e., apoptosis and necrosis) (9, 10).Like many other A–B toxins that mediate their own delivery into cells, high-resolution structures of the enzymic A domains (11–13) and the receptor-binding portion of the B domains of glucosylating toxin family members are known (14, 15), whereas the structure and mechanism of the pore-forming translocation domain remains poorly characterized. These interconnected processes have been proposed to be mediated by the central ∼1,000-aa D domain (i.e., amino acids 801–1,850); however, with the absence of any structural information for this domain in either the prepore or pore state, no framework exists for resolving the functional determinants for this large domain that govern pore formation and translocation. It is well established that in response to acidic pH, the D domain undergoes a conformational change that results in the formation of ion-conductive pores in both biological membranes and artificial lipid bilayers (16, 17).It has been hypothesized that the cluster of 172 hydrophobic, highly conserved amino acids in the middle of the translocation domain (i.e., residues 958–1,130 in TcdA and 956–1,128 in TcdB) comprised some, if not all, of the segments that form the translocation pore (18). Demonstrating this, however, has been challenging, in large part due to difficulties associated with manipulating clostridial toxin genes at the genetic level. The recent availability of clones of both TcdA and TcdB in Bacillus megaterium expression plasmids, which enable the high-level production of stably folded toxin, has facilitated research in this direction (19, 20); however, studies specifically addressing the structure and function of the translocation domain have been limited to large-fragment deletions to probe function (21, 22).In the present study, we set out initially with the goal of identifying the determinants of pore formation and translocation through a comprehensive mutagenesis study using the B. megaterium platform. We discovered very early in this pursuit that site-specific mutagenesis of the inherently AT-rich toxin sequence (i.e., G + C = 27%) using the B. megaterium system was laborious and inefficient. To address this, we generated a GC-enriched copy of TcdB (i.e., G + C = 45%) with codons optimized for Escherichia coli expression, which allowed us to perform high throughput probing of the translocation domain. We identify several single point mutations clustering to within the hydrophobic region of the delivery domain that result in major defects in pore formation and translocation. We report the unexpected similarity of the identified pore-forming region to that of the translocation domain of DT and use these data en bloc to propose an α-helical model for the translocation pore of TcdB and homologous pathogenic toxins.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

A ribosome-inactivating protein in a Drosophila defensive symbiont

Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense.Symbiosis is now recognized to be a key driver of evolutionary novelty and complexity (1, 2), and symbioses between microbes and multicellular hosts are understood as essential to the health and success of diverse lineages, from plants to humans (3). Insects, in particular, have widespread associations with symbiotic bacteria, with most insect species infected by maternally transmitted endosymbionts (4, 5). Although many insect symbionts perform roles essential for host survival, such as supplementing nutrition, others are facultative and not strictly required by their hosts. These facultative symbionts have evolved diverse and intriguing strategies to maintain themselves in host populations despite loss from imperfect maternal transmission and metabolic costs to the host. These range from manipulating host reproduction to increase their own transmission (6, 7), such as by killing male hosts, to providing context-dependent fitness benefits (8). Recently, it has become clear that different insect endosymbionts have independently evolved to protect their hosts against diverse natural enemies that so far include pathogenic fungi (9), RNA viruses (10, 11), parasitoid wasps (12), parasitic nematodes (13), and predatory spiders (14, 15). This suggests that defense might be a common aspect of many insect symbioses and demonstrates that symbionts can serve as dynamic and heritable sources of protection against natural enemies (8).Despite a growing appreciation of the importance of symbiont-mediated defense in insects, key questions remain. Most demonstrations of defense have been under laboratory conditions, and the importance of symbiont-mediated protection in natural systems is unclear in most cases (16). At the same time, the proximate causes of defense are largely unknown, although recent studies have provided some intriguing early insights: A Pseudomonas symbiont of rove beetles produces a polyketide toxin thought to deter predation by spiders (14), Streptomyces symbionts of beewolves produce antibiotics to protect the host from fungal infection (17), and bacteriophages encoding putative toxins are required for Hamiltonella defensa to protect its aphid host from parasitic wasps (18), whereas the causes of other naturally occurring defensive symbioses are unresolved. From an applied perspective, the ongoing goal of exploiting insect symbioses to arrest disease transmission to humans from insect vectors (19) makes a deeper understanding of the factors contributing to ecologically relevant and evolutionarily durable defensive symbioses urgently needed.Here, we investigate the mechanism underlying one of the most striking examples of an ecologically important defensive symbiosis. Drosophila neotestacea is a woodland fly that is widespread across North America and is commonly parasitized by the nematode Howardula aoronymphium. Infection normally sterilizes flies (20); however, when flies harbor a strain of the inherited symbiont Spiroplasma—a Gram-positive bacterium in the class Mollicutes—they remarkably tolerate Howardula infection without loss of fecundity, and infection intensity is substantially reduced (13). The benefit conferred by this protection lends a substantial selective advantage to Spiroplasma-infected flies and has led to Spiroplasma’s recent spread across North America, with symbiont-infected flies rapidly replacing uninfected ones (21). Spiroplasma is a diverse and widespread lineage of arthropod-associated bacteria that can be commensal, pathogenic, or mutualistic (22). Maternal transmission has arisen numerous times in Spiroplasma, including strains that are well known as male-killers (22). In addition to defense against nematodes in D. neotestacea, other strains of Spiroplasma have recently been shown to protect flies and aphids against parasitic wasps and pathogenic fungi, respectively (23–25), but in no case is the mechanism of defense understood.In theory, there are multiple avenues by which a symbiont may protect its host that include competing with parasites for limiting resources, priming host immunity, or producing factors to directly attack parasites (26). We previously assessed these possibilities in the defensive Spiroplasma from D. neotestacea (27); our findings best supported a role for toxins in defense, with Spiroplasma encoding a highly expressed putative ribosome-inactivating protein (RIP). RIPs are widespread across plants and some bacteria and include well-known plant toxins of particular human concern such as ricin, as well as important virulence factors in human toxigenic strains of Escherichia coli and Shigella (28, 29). RIPs characteristically exert their cytotoxic effects through depurination of eukaryotic 28S ribosomal RNAs (rRNAs) at a highly conserved adenine in the α-sarcin/ricin loop (SRL) of the rRNA by cleaving the N-glycosidic bond between the rRNA backbone and adenine (30, 31). The proliferation of RIPs across different lineages implies functional significance, but their ecological roles are unclear, although they often appear to have antiviral or other defensive roles (29, 32). Here, we find that Spiroplasma expresses a functional RIP distinct from previously characterized toxins that appears to specifically affect Howardula rRNA in flies coinfected with Spiroplasma and Howardula. This work suggests the mechanisms used in defensive associations to protect the host from disease as well as intriguing ecological roles for RIPs in a tripartite defensive symbiosis.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1–DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.Eukaryotic translation elongation factor 2 (eEF2) is a highly conserved protein and essential for protein biosynthesis. EEF2 enables peptide-chain elongation by translocating the peptide–tRNA complex from the A- to the P-site of the ribosome (1, 2). The diphthamide modification at His715 of human eEF2 (or at the corresponding position in other species) is conserved in all eukaryotes (3) and in archaeal counterparts. It is generated by proteins that are encoded by seven genes (4). Proteins encoded by dipthamide biosynthesis protein (DPH)1, DPH2, DPH3, and DPH4 (DNAJC24) attach a 3-amino-3-carboxypropyl (ACP) group to eEF2. This intermediate is converted by the methyltransferase DPH5 to diphthine, which is subsequently amidated to diphthamide by DPH6 and DPH7 (5).Diphthamide synthesis was previously described in yeast and other eukaryotes (4–6). However, the “complete picture” is (with the exception of the yeast pathway) to a large portion is composed of observations made in different cell types on single genes. Many reports related to diphthamide synthesis of mammalian cells describe “partial knockouts” and “partial phenotypes” (i.e., reduced levels but not complete loss of diphthamide modification or toxin sensitivities) (7–9). Because mammalian genomes are more complex than that of yeast, carrying extendend gene families, mammalian cells may compensate—at least to some degree—functional loss of genes that may be unique and essential in yeast. If and to what degree mammalian cells can compensate a partial or complete loss of DPH gene functionality (and with what consequences) is unknown to date.So far, the function of diphthamide on eEF2 also remained rather elusive. Reports indicate that it contributes to translation fidelity (10–13). On the other hand, DPH genes or eEF2 can be mutated to prevent diphthamide attachment, yet cells carrying such mutations are viable (5, 11, 14, 15). Animals with heterozygous DPH knockouts (DPHko) can be generated, but homozygous DPH1ko, DPH3ko, and DPH4ko are embryonic lethal (13, 16–18). Because these studies are based on inactivation of individual genes, it is difficult to discriminate between phenotypes caused by gene loss and phenotypes as a consequence of loss of diphthamide.Diphthamide-modified eEF2 is the target of ADP ribosylating toxins, including Pseudomonas exotoxin A (PE) and diphtheria toxin (DT) (19). These bacterial proteins enter cells and catalyze ADP ribosylation of diphthamide using nictotinamide adenine dinucleotide (NAD) as substrate (20, 21). This inactivates eEF2, arrests protein synthesis, and kills (14). Tumor-targeted PE and DT derivatives are applied in cancer therapies (22–28) and their efficacy depends on toxin sensitivity of target cells. Therefore, information about factors (and their relative contributions) that influences cellular sensitivities toward diphthamide-modifying toxins may predict therapy responses. For example, alterations in OVCA1 (human DPH1) were described for ovarian cancers (16, 29), yet it is not known if and to what degree such alterations would affect sensitivities of tumor cells toward PE-derived drugs.Here we describe MCF7 breast cancer cell line derivatives with heterozygous or complete DPH gene inactivations. These cells are applied to analyze the contributions of individual DPHs not only to diphthamide synthesis and toxin sensitivity, but also to address gene dose effects. Because the set of knockout cell lines is derived from the same parent cell and provides loss of diphthamide as common consequence of inactivation of different genes, these cells can also shed light on the biological relevance of the diphthamide modification.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.