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.     

Visualization of the type III secretion sorting platform of Shigella flexneri

Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.Type III secretion systems (T3SSs) are essential virulence determinants for many Gram-negative pathogens. The injectisome, also known as the needle complex, is the central T3SS machine required to inject effector proteins from the bacterium into eukaryotic host cells (1, 2). The injectisome has three major components: an extracellular needle, a basal body, and a cytoplasmic complex (3). Contact with a host cell membrane triggers activation of the injectisome and the insertion of a translocon pore into the target cell membrane. The entire complex then serves as a conduit for direct translocation of effectors (1, 2). Assembly of a functional T3SS requires recognition and sorting of specific secretion substrates in a well-defined order by the cytoplasmic complex (4, 5). Furthermore, genes encoding the cytoplasmic complex are regulated by physical and environmental signals (6), providing temporal control of the injection of effector proteins and thereby optimizing invasion and virulence.Significant progress has been made in elucidating T3SS structures from many different bacteria (7, 8). 3D reconstructions of purified injectisomes from Salmonella and Shigella, together with the atomic structures of major basal body proteins, have provided a detailed view of basal body architecture (9, 10). Recent in situ structures of injectisomes from Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica revealed an export gate and the structural flexibility of the basal body (11, 12). Unfortunately, these in situ structures from intact bacteria (11, 12) did not reveal any evident densities related to the proposed model of the cytoplasmic complex (8, 13).The flagellar C ring is the cytoplasmic complex in evolutionarily related flagellar systems. It is composed of flagellar proteins FliG, FliM, and FliN and plays an essential role in flagellar assembly, rotation, and switching (14). Large drum-shaped structures of the flagellar C ring have been determined in both purified basal bodies (15, 16) and in situ motors (17–19). Similarly, electron microscopy analysis in Shigella indicated that the Spa33 protein (a homolog of the flagellar proteins FliN and FliM) is localized beneath the basal body via interactions with MxiG and MxiJ and is an essential component of the putative C ring (20). Recent experimental evidence suggests that the putative C ring provides a sorting platform for the recognition and secretion of the substrates in S. enterica (5). This sorting platform consists of three proteins, SpaO, OrgA, and OrgB, which are highly conserved among other T3SSs (21) (SI Appendix, Table S1). Despite its critical roles, little is still known about the structure and assembly of the cytoplasmic sorting platform in T3SS. In this study, we choose S. flexneri as a model system to study the intact T3SS machine and its cytoplasmic complex, mainly because a wealth of structural, biochemical, and functional information is available for the S. flexneri T3SS (22).     

Marker for type VI secretion system effectors

Bacteria use diverse mechanisms to kill, manipulate, and compete with other cells. The recently discovered type VI secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Using comparative proteomics, we identified two previously unidentified T6SS effectors that contained a conserved motif. Bioinformatic analyses revealed that this N-terminal motif, named MIX (marker for type six effectors), is found in numerous polymorphic bacterial proteins that are primarily located in the T6SS genome neighborhood. We demonstrate that several MIX-containing proteins are T6SS effectors and that they are not required for T6SS activity. Thus, we propose that MIX-containing proteins are T6SS effectors. Our findings allow for the identification of numerous uncharacterized T6SS effectors that will undoubtedly lead to the discovery of new biological mechanisms.The type VI secretion system (T6SS), a recently discovered protein secretion machinery (1), is a tool used by Gram-negative bacteria to inject effector proteins into recipient cells (2). During the type VI secretion process, an intracellular tube complex composed of hexameric rings of haemolysin coregulated proteins (Hcp) capped with a trimer of valine-glycine repeat protein G (VgrG) and a proline-alanine-alanine-arginine (PAAR) repeat-containing protein is surrounded by a sheath made of VipA/VipB heterodimers (also known as TssB/TssC). Upon an extracellular signal, the sheath contracts, leading to secretion of the tube complex into an adjacent target cell (2–4). Multiple T6SSs can be encoded within a single bacterial genome (5), and each T6SS can have more than one cognate Hcp, VgrG, or PAAR repeat-containing protein (4).T6SS effectors are predicted to be loaded onto the tube complex by several distinct mechanisms: as toxin domains fused to VgrG or PAAR repeat-containing proteins, as proteins that bind the inner surface of the Hcp tube, or as proteins that interact with VgrG or PAAR repeat-containing proteins (2). Two T6SS effector families have been characterized: peptidoglycan hydrolases (6) and phospholipases (7). Additional effector activities, such as nucleases (8), actin cross-linking (9), ADP ribosylation (10), and pore-forming (11), have also been described. Notably, T6SS effectors with antibacterial activities are paired with a cognate immunity protein encoded downstream of the effector gene to prevent self-intoxication (6, 12).We have recently described an antibacterial activity for T6SS1 of the marine bacterium Vibrio parahaemolyticus, a leading cause of gastroenteritis (13), and identified the environmental conditions required for its activation (14). Surprisingly, no known T6SS effectors are found in the genome of the V. parahaemolyticus RIMD 2210633 isolate, suggesting this strain harbors previously unidentified T6SS effectors.Here, we set out to identify V. parahaemolyticus T6SS1 effectors that mediate its antibacterial activity. Using comparative proteomics, we identified several T6SS effectors and their cognate immunity proteins. Remarkably, we found a motif named MIX (marker for type six effectors) that was shared by two of the newly identified effectors. We hypothesized and subsequently showed that this motif is found in numerous bacterial proteins with diverse predicted or established bacteriocidal and virulence activities, among them several confirmed T6SS effectors. Thus, we propose that proteins containing the MIX motif are polymorphic T6SS effectors.     

A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii

Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells specialized in the elimination of competing bacteria.Antibiotic-resistant bacteria that cause hospital-acquired infections are a mounting concern for health care systems globally (1). Multidrug-resistant (MDR) Acinetobacter baumannii is emerging as a frequent cause of difficult-to-treat nosocomial infections, and some isolates are resistant to all clinically relevant antibiotics (2, 3). A. baumannii is often isolated from polymicrobial infections and therefore spends at least a part of its time competing with other bacteria (4). Antagonistic interactions between bacteria manifest in a variety of different ways (5), and the type VI secretion system (T6SS) is a potent weapon used by many Gram-negative bacteria to kill competitors (6–8). The multicomponent T6SS apparatus facilitates a dynamic contact-dependent injection of toxic effector proteins into prey cells (9, 10), and expression of cognate immunity proteins prevents self-inflicted intoxication (9, 11). The T6SS is composed of several conserved proteins involved in the formation of the secretory apparatus (12, 13). One of these components, hemolysin-coregulated protein (Hcp), forms hexameric tubule structures that are robustly secreted to the culture supernatants in bacteria with an active T6SS, allowing it to be used as a molecular marker for T6SS activity (6, 14).T6SS is a dynamic apparatus (15). Its biogenesis follows energetically costly cycles of assembly/disassembly, and therefore, in most bacteria, T6SS appears to be exquisitely regulated. T6SS is silenced in most strains and only activated under specific conditions, such as an attack from another bacterium or in environments leading to membrane perturbations (16–19). Many Acinetobacter spp. encode the genes for a T6SS, including Acinetobacter noscomialis and Acinetobacter baylyi, which possess a constitutively active antibacterial T6SS (20–24). A. baumannii strains have been shown by us and others to secrete Hcp (21, 25), but to our knowledge a T6SS-dependent phenotype has not been ascribed to this species. Furthermore, our previous results showed that Hcp secretion is highly variable between A. baumannii strains, with some isolates carrying an inactive system (21). The precise regulatory mechanism(s) underlying T6SS suppression in some A. baumannii is unknown.Here, we show that a large resistance plasmid of A. baumannii functions to repress the T6SS by encoding negative regulators of its activity. Analysis of colonies from a clinical isolate showed that the plasmid is readily lost in a subset of the population. This leads to the activation of the T6SS, which imparts the ability to kill other bacteria, with the simultaneous loss of antibiotic resistance. We propose that the differentiation into T6SS+ MDR– and T6SS– MDR+ phenotypes may constitute a novel survival strategy of this organism.     

Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae

Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host–pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis.Plants evoke resistance against invading bacteria using plasma membrane-localized pattern recognition receptors (PRRs) to detect the presence of pathogen-associated molecular patterns (PAMPs) in the extracellular space (1). Activation of PRRs by PAMPs results in numerous defense responses that limit bacterial growth (1). However, the actual mechanisms by which plants suppress virulence and restrict bacterial growth remain unclear. Pseudomonas syringae is a model bacterial pathogen that infects a wide range of economically important crops as well as the laboratory model plant Arabidopsis (2). P. syringae uses several different virulence strategies to suppress host defenses, including a type III secretion system (T3SS) that secretes up to 30 effector proteins into plant cells (3, 4). Many effectors function to suppress PRR-induced signaling, thereby allowing the bacteria to avoid detection and proliferate (4). Mutants of P. syringae lacking a functional T3SS are not fully virulent, demonstrating that this system is essential for a successful infection (5, 6). Moreover, recent studies have revealed that PAMP-triggered immunity (PTI) leads to a restriction in the delivery of type III effectors into host cells, suggesting that plants possess an unknown mechanism(s) to block type III secretion (7, 8).Despite the critical role of the T3SS in P. syringae virulence, T3SS structural components and effectors are not constitutively present but are produced at the onset of infection (9, 10). Early attempts to identify plant signals perceived by P. syringae revealed that synthetic medium mimicking the plant apoplast, namely a minimal nutrient medium with acidic pH and including a sugar such as fructose, is capable of inducing T3SS-associated genes (9–12). However, in some instances expression of the T3SS was higher in planta than in synthetic medium, indicating that additional plant-derived factors likely were required for full induction (10, 12). These results imply the presence of plant-derived signal(s) that induce the T3SS, and various signals have been proposed to be capable of inducing the T3SS in different plant pathogenic bacteria based largely on in vitro experiments (10, 12–17). However, whether any of these signals affect the biological outcome of the host–pathogen interaction remains speculative because of the lack of genetic mutants altering the abundance of these chemical signals in the host.In the present work, we identify host chemical signals that DC3000 uses to switch to its virulence program and demonstrate that this recognition event plays an important role in a successful infection. The identification of an Arabidopsis mutant, MAPK phosphatase 1 (mkp1), in which the delivery of the P. syringae pv tomato DC3000 effector is suppressed, provided an important genetic model for investigating the basis for T3SS induction. Using a metabolomics comparison of mutant and WT plant exudates, we identified several plant-derived metabolites that are present at lower levels in mkp1 and induce the T3SS in DC3000. The biological significance of these compounds was demonstrated by showing that reintroducing these T3SS-inducing metabolites can overcome both the suppression of effector delivery and the enhanced resistance in mkp1 plants. Furthermore, the addition of these metabolites also can overcome enhanced resistance induced in plants pretreated with PAMPs. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the levels of these host-derived signals contribute to susceptibility or resistance.     

Generation of reactive oxygen species by lethal attacks from competing microbes

Whether antibiotics induce the production of reactive oxygen species (ROS) that contribute to cell death is an important yet controversial topic. Here, we report that lethal attacks from bacterial and viral species also result in ROS production in target cells. Using soxS as an ROS reporter, we found soxS was highly induced in Escherichia coli exposed to various forms of attacks mediated by the type VI secretion system (T6SS), P1vir phage, and polymyxin B. Using a fluorescence ROS probe, we found enhanced ROS levels correlate with induced soxS in E. coli expressing a toxic T6SS antibacterial effector and in E. coli treated with P1vir phage or polymyxin B. We conclude that both contact-dependent and contact-independent interactions with aggressive competing bacterial species and viruses can induce production of ROS in E. coli target cells.Microbial species exist predominantly in complex communities in the natural environment and animal hosts. To survive in a multispecies environment, bacteria have developed various strategies to compete with other species. For example, some bacteria can exert long-range inhibitory effects by secreting diffusible molecules, such as antibiotics, bacteriocins, and H₂O₂ (1), whereas others require direct cell-to-cell contact to kill nearby organisms (2, 3). One such contact-dependent inhibitory system is the type VI secretion system (T6SS), a protein translocating nanomachine expressed by many Gram-negative bacterial pathogens that can kill both bacterial and eukaryotic cells (3–5). Structurally analogous to an inverted bacteriophage tail, the T6SS delivers effectors into target cells by using a contractile sheath to propel an inner tube out of the producer cell and into nearby target cells. The inner tube (composed of Hcp protein) is thought to carry toxic effector proteins within its lumen or on its tip, which is decorated with VgrG and PAAR proteins (4, 6, 7). Given that some cells can detect T6SS attack but not suffer any measurable loss in viability (8, 9), it would seem that cell killing is likely due to the toxicity of effectors rather than membrane disruptions caused by insertion of the spear-like VgrG/PAAR/Hcp tube complex. T6SS-dependent effectors can attack a number of essential cellular targets, including the cell wall (10, 11), membranes (11, 12), and nucleic acids (13), and thus can mimic the actions of antibiotics and bacteriocins. As a model prey or target organism, Escherichia coli can be killed by the T6SS activities of a number of bacteria including Vibrio cholerae (14), Pseudomonas aeruginosa (10, 15), and Acinetobacter baylyi ADP1 (7).Collins and coworkers (16–18) have reported that antibiotic treatment of E. coli elicits the production of reactive oxygen species (ROS) resulting from a series of events involving perturbation of the central metabolic pathway, NADPH depletion, and the Fenton reaction. ROS can cause lethal damage to DNA, lipid, and proteins (19, 20) and thus can contribute to cell death in combination with the deleterious effects of antibiotics on their primary targets. The idea that antibiotics kill bacterial cells, in part, through the action of ROS has been supported by a number of follow-up studies (18, 21–23) but has also been challenged by others as a result of observations contradictory to a model where ROS is the sole mediator of antibiotic lethality (24–26). These observations include the fact that antibiotics kill under anaerobic conditions, oxidation of the hydroxyphenyl fluorescein fluorescence dye used to measure ROS levels is nonspecific, and the extracellular level of H₂O₂ is not elevated by antibiotic treatment (24, 26). To address these concerns, Dwyer et al. (27) used a panel of ROS-detection fluorescence dyes, a defined growth medium under stringent anaerobic conditions, and an in vivo H₂O₂ enzymatic assay to study the effects of antibiotics on cells. The results further support that antibiotics induce ROS generation, which contributes to the efficacy of antibiotics in addition to their primary lethal actions (18, 27, 28).     

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.It is well accepted that direct virus neutralization is an important element of antibody-mediated protection against HIV-1 (refs. 1–6 and reviewed in ref. 7). In contrast, less is known about the role of Fc-mediated effector function in the control of HIV-1, although four lines of evidence signal its importance. First, studies in HIV-1–infected people (8–14) and in macaques infected with simian immunodeficiency virus (15, 16) consistently show an inverse correlation between Fc-mediated effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) (8, 9) or antibody-dependent cell-mediated viral inhibition (ADCVI), and viral loads or decreased disease progression (17). Second, vaccine-elicited protection both in nonhuman primates (18–21) and in a subset of human subjects in the Vax-004 trial (22) correlates with Fc-mediated effector function often observed in the absence of detectable neutralizing antibodies (18–21). Similarly, there was an inverse relationship between acquisition of HIV-1 and ADCC in the RV144 trial for a subset of subjects who had low to moderate IgA anti-gp120 titers (23). Third, breast milk IgG ADCC responses to gp120 but not to virus neutralization correlated with reduced perinatal transmission of HIV-1 (24). Fourth, passive immunization studies in nonhuman primates (25, 26) showed that abrogation of Fc-mediated effector function diminished the sterilizing protection afforded by the neutralizing mAb b12. These compelling studies show that neutralization alone significantly protects against a simian-human immunodeficiency virus challenge and that Fc-mediated effector function augments this effect. Taken together, these four lines of investigation strongly suggest that Fc-mediated effector function in addition to neutralization contributes to antibody-mediated protection against HIV-1. Thus, it is important to determine the precise relationships among antibody specificity, neutralization, and Fc-mediated effector function in protection against HIV-1.In this report, we probe these relationships using a panel of human mAbs that recognize transitional epitopes exposed during the earliest stage of viral entry, the interaction of gp120 with CD4. Our studies deliberately focus on antibody responses to epitopes that become exposed during viral entry because passive immunization studies indicate that an antibody has at most a 24-h window to block transmission (ref. 27; reviewed in ref. 28). Thus, transmission-blocking antibodies must block infection by direct neutralization of HIV-1, by Fc-mediated killing of nascently infected cells, or both. Although these two effector functions often are coincident for a given mAb specificity (29, 30), they can be dissociated because nonneutralizing epitopes on both gp120 (12, 31) and gp41 (32) can be ADCC targets. In this report, we probe the relationships among antibody specificity, ADCC, and neutralization using a panel of human mAbs that recognize transitional epitopes exposed on target cells during viral entry.     

T cell-intrinsic S1PR1 regulates endogenous effector T-cell egress dynamics from lymph nodes during infection

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1^−/− effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1^−/− T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1^−/− effector T cells failed to enter the sinuses. We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.An effective immune response depends on the large-scale, but carefully regulated, migration of T cells within and between lymphoid and peripheral tissues. This migration is tightly regulated by several factors, including the highly organized secondary lymphoid structure and the cellular expression of chemokine receptors and compartmentalized secretion of their cognate ligands (1). This balance between the anatomy and the ordered expression of cell surface and soluble proteins dictates the exquisite choreography of T-cell migration, and visualizing these dynamics of T-cell behavior in situ within the lymph nodes (LNs) is essential for understanding the mechanisms that mediate the generation of a productive antimicrobial or antitumoral immune response (1, 2). However, our understanding of the factors that regulate the anatomical program followed by endogenous antigen-specific effector T cells after an infection remains incomplete, especially with respect to the mechanisms that regulate egress kinetics of effector T cells from LN (2, 3).T-cell migration, even at steady state, is a highly regulated process (4). T-cell entry into the LN is controlled by G protein-coupled receptors (GPCRs) (3) such as CC chemokine receptor 7 (CCR7), which is also critical for the localization and retention of T cells within the LN paracortex (5, 6). Egress of naive T cells from the LN via the lymphatic vessels is regulated by the GPCR sphingosine-1-phosphate receptor-1 (S1PR1) (3) and adhesion molecules (4). S1PR1 is among four other GPCRs that bind to sphingosine-1-phosphate (S1P) with high affinity. S1PR1 is abundantly expressed in different cell types and tissues, including immune cells and endothelial cells (7). In addition to mediating lymphocyte egress, binding of S1P to S1PR1 and other receptors (S1PR2 to -5) on the cell surface initiates several signaling cascades that affect the functioning of many organ systems and control a multitude of biological functions, including inflammation, cell survival and differentiation, cardiovascular function, and vascular permeability (8).Several recent studies have investigated the mechanisms by which S1PR1 regulates naive T-cell egress (9–14). Several groups have used FTY720 (Fingolimod) to investigate the role of S1PR1 in vivo. However, FTY720 is a broad modulator of four different S1P receptors (S1PR1 and S1PR3 to -5) and is a potent receptor agonist that initially activates the receptors but subsequently induces the internalization and degradation of the S1P receptors and consequently is considered a functional antagonist. Indeed, administration of FTY720 in mice blocks naive T-cell egress from the thymus and LN (15) and peripheral homing of effector T cells (16). However, it is not clear whether FTY720 exerts its effect primarily by targeting the endothelial cells or T cells although a recent study favored the idea that FTY720 exerts its function by targeting T cells (14). Nevertheless, studies that favor a T-cell intrinsic mechanism by which S1PR1 regulates naive T-cell egress used S1PR1^−/− T cells to demonstrate that S1PR1-deficient naive T cells failed to exit the thymus (17, 18) and LN (11), which mimicked the results obtained by using FTY720. However, S1PR1 is also abundantly expressed on endothelial cells, where it regulates lymphatic vascular permeability (7, 19, 20). Studies using S1PR1-specific agonists (21) and antagonists (9, 22) have implicated a more critical role of lymphatic S1PR1 in regulating naive lymphocyte egress, evidently by sealing the lymphatic endothelial barrier and disallowing naive T-cell egress from LN and thymus. These studies have been instrumental in improving our understanding of how S1PR1 controls naive lymphocyte migration. However, the mechanisms that regulate the dynamics of effector T-cell egress during an infection are less understood (3).The exact intranodal localization, exit points, and mechanisms that regulate the complex process of effector T-cell egress from the draining lymph node (dLN) after infection and inflammatory conditions are not well understood. Moreover, it is not known, after T-cell activation after an infection, when LN retention signals such as CCR7 and its ligands are down-regulated (23), whether the effector T cells are still dependent on S1PR1 for egress. Furthermore, in an inflammatory milieu after infection, when S1P levels are likely altered (8, 20, 24), it remains to be determined what are the roles of T cell-intrinsic or stromal cell-intrinsic expression of S1PR1 in regulating effector T-cell trafficking. Therefore, in the current study, we addressed these unanswered questions by disrupting S1PR1 signals specifically and temporally in endogenous effector CD8 T cells after infection to demonstrate the mechanisms that regulate the dynamics of endogenous effector CD8 T-cell egress after infection.     

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA₁) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA₁ activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA₁ domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA₁ activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization.Microbial pathogens have evolved numerous ways to subvert and exploit normal host cell processes and to cause disease. Intravacuolar pathogens use specialized translocation devices such as type IV secretion systems (T4SS) to deliver virulence proteins, so-called effectors, across the bacterial and host cell membrane into the cytosol of the infected cell (1–3). Many of the translocated effectors studied to date alter cellular events such as vesicle trafficking, apoptosis, autophagy, protein ubiquitylation, or protein synthesis, among others, thereby creating conditions that support intracellular survival and replication of the microbe (4, 5). Bacteria with a nonfunctional T4SS are often avirulent and degraded along the endolysosomal pathway, thus underscoring the importance of translocated effectors for microbial pathogenesis.Although T4SS-mediated effector translocation may be a convenient way for pathogens to manipulate host cells from within the safety of their membrane-enclosed compartment, it also creates a challenging dilemma: how can the bacteria ensure that their translocated effectors reach the correct host cell target for manipulation, and how can they prevent them from indiscriminately affecting bystander organelles or proteins that may otherwise be beneficial for intracellular survival and replication of the microbe? It is reasonable to expect that regulatory mechanisms have evolved that restrain the catalytic activity of effectors. Although detailed insight into these processes is scarce, an emerging theme among effectors is that their enzymatic activity is functionally coupled to their interaction with a particular host factor. For example, SseJ from Salmonella enterica serovar Typhimurium displays glycerophospholipid-cholesterol acyltransferase activity only on binding to the active GTPases RhoA, RhoB, or RhoC (6–8). Likewise, Pseudomonas aeruginosa ExoU requires mono- or poly-ubiquitinated proteins for the activation of its phospholipase A2 (PLA₂) domain (9), whereas Yersinia YpkA exhibits kinase activity only in the presence of host cell actin (10). Exactly how binding to host ligands results in the activation of these translocated effectors remains unclear because no structural information for these protein complexes is available.VipD is a T4SS-translocated substrate of Legionella pneumophila, the causative agent of a potentially fatal pneumonia known as Legionnaires'' disease, and another example of an effector whose catalytic activity depends on the presence of a host factor (11–14). Following uptake by human alveolar macrophages, L. pneumophila translocates VipD together with more than 250 other effector proteins through its Dot/Icm T4SS into the host cell cytoplasm (15). These effectors act on numerous host processes to mediate evasion of the endolysosomal compartment and to establish a Legionella-containing vacuole (LCV) that supports bacterial growth (16). Although the precise biological role of most L. pneumophila effectors remains unclear, we recently showed that VipD is important for endosomal avoidance by LCVs. The protein localizes to endosomes presumably by binding to the small GTPases Rab5 or Rab22, key regulators of endosomal function (13, 14). Rab GTPase binding to the C-terminal domain of VipD triggers robust phospholipase A1 (PLA₁) activity within the N-terminal domain, resulting in the removal of phosphatidylinositol 3-phosphate [PI(3)P] and potentially other lipids from endosomal membranes (14). Without PI(3)P, endosomal markers such as early endosomal antigen 1 (EEA1) are lost from these membranes, most likely rendering the endosomal compartment fusion incompetent (17). L. pneumophila mutants lacking vipD are attenuated in avoiding endosomal fusion, and their LCVs acquire the endosomal marker Rab5 more frequently than LCVs containing the parental strain producing VipD (14). Thus, by coupling PLA₁ activity to Rab5 binding, the catalytic activity of VipD is directed specifically against the endosomal compartment without visibly affecting neighboring cell organelles.VipD was originally identified in a screen for L. pneumophila effectors that interfere with the vacuolar sorting pathway in yeast (11). The N-terminal half of VipD possesses high homology to patatin, a lipid acyl hydrolase present in the potato tuber (12, 13). Analogous to other patatin-like proteins, VipD harbors a conserved serine lipase motif Gly-x-Ser-x-Gly (x = any amino acid) as part of a Ser-Asp catalytic dyad that, together with two consecutive glycine residues (Asp-Gly-Gly motif), is expected to stabilize the oxyanion intermediate during the acyl chain cleavage (13). Mutation of these conserved catalytic residues in VipD results in loss of PLA₁ activity (14), confirming their role in substrate hydrolysis.The recently reported crystal structure of VipD confirmed the predicted bimodular organization (13) and, in addition, revealed a surface loop, called “lid” in other phospholipases, that shields the entry to the catalytic site. The inhibitory lid may explain why purified recombinant VipD alone exhibits little or no PLA₁ activity in vitro. However, given that binding of Rab5 or Rab22 to VipD activates the PLA₁ activity within the N-terminal region (14), we wondered if and how this binding event causes the inhibitory lid to be removed to render the active site substrate accessible.Using an integrative approach involving X-ray crystallography, molecular dynamics, biochemistry, and cellular imaging, we now deciphered at a molecular level the mechanism that stimulates the intrinsic PLA₁ activity of VipD and determined the underlying specificity for the VipD–Rab5 interaction and endosomal targeting.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread

Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laser-scanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.Toxoplasma gondii infects around a third of humans worldwide and is widely dispersed in other warm-blooded hosts. Although clinical manifestations in the brain, eye, and developing fetus receive the most attention, T. gondii is an oral pathogen and first enters the body and establishes infection in the small intestine. Infection follows consumption of cyst-containing meat or oocyst-contaminated water and produce and is associated with the development of small intestinal pathology in a variety of nonhuman hosts (1). Most notably, experimental infection of C57BL/6 mice by the oral route results in an inflammation of the small intestine that shares immunological features with inflammatory bowel disease (2). This model is useful to further our understanding of host–pathogen interactions in the intestine and of common mechanisms underpinning the development of inflammatory bowel disease (3). Nevertheless, we have limited understanding of how and in which cells infection is established in the intestine, the extent to which the parasite replicates and spreads within the intestine, and how these factors contribute to the development of pathology (2, 4–9). The ability to label living parasites fluorescently and track them in the tissues of infected hosts provides an important tool for investigating these questions (10–14).Starting in the small intestine, T. gondii must travel long distances and surmount a variety of biological barriers to establish chronic infection in the brain. These barriers include the mucus, the intestinal epithelium, and the blood–brain barrier (7, 15). Cells of the immune system are often highly motile and represent attractive transport vessels for pathogens seeking to reach and enter tissues while being protected from the external environment. Consequently, recent studies have focused on the role of immune cells in transporting parasites between tissues (4, 16–23). For example, cluster of differentiation 11b-positive (CD11b⁺) cells have been implicated in the dissemination of T. gondii through the blood and across the blood–brain barrier (4, 19). Following oral infection, it is thought that the initial invasion or traversal of intestinal epithelial cells by ingested parasites is followed by parasite replication in tissue and the transport by host cells to other tissues. However, our understanding of how the parasite enters and disseminates through the intestine itself and of the role played by immune cell populations in this initial phase of infection is extremely limited.Two-photon microscopy provides important spatial and dynamic information to further our understanding of how pathogens interact with their hosts in complex natural tissue environments (10). However, such techniques rarely have been applied to the intestine, particularly in the context of infection (14, 24–28). Here we used a physiologically relevant oral infection model in conjunction with two-photon microscopy to reveal that neutrophils in the lumen of the small intestine are motile reservoirs of live T. gondii. Our findings implicate these cells in an unexpected luminal pathway for the spread of infection.     

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

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

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.     

Dimer recognition and secretion by the ESX secretion system in Bacillus subtilis

Protein secretion typically involves translocation of unfolded polypeptides or transport of monomeric folded proteins. Here we provide, to our knowledge, the first experimental evidence for secretion of an intact multimeric complex requiring a signal formed by both members of the complex. Using systematic mutagenesis of a substrate involved in early secretory antigen 6 kDa (ESX) secretion in Bacillus subtilis, we demonstrate that export of the substrate requires two independent motifs. Using mixed dimers, we show that these motifs must form a composite secretion signal in which one motif is contributed by each subunit of the dimer. Finally, through targeted crosslinking we show that the dimer formed in the cell is likely secreted as a single unit. We discuss implications of this substrate recognition mechanism for the biogenesis and quality control of secretion substrates and describe its likely conservation across ESX systems.Protein secretion is critical for protein targeting in any living cell and for its communication with the environment. Bacteria use a wide range of secretion mechanisms to export proteins out of the cytoplasm. Signals for secretion are most commonly primary amino acid sequences, but in some cases also may be formed through interacting surfaces of a substrate and its delivery effector. Some secretion systems unfold their substrates to translocate them across the membrane and cell wall. Other systems export folded proteins, sometimes in complex with bound cofactors. For example, the general secretory machinery (Sec) denatures the tertiary and secondary structure of its substrates to thread the polypeptide through the narrow opening of the integral membrane translocon complex, SecYEG (1). Type III secretion system (T3SS) machinery is thought to unfold the tertiary structure of its substrates, while preserving the secondary structure elements for the substrate recognition (2, 3). In contrast, the twin-arginine transport (Tat) system exports folded substrates (4) and is hypothesized to be able to translocate protein oligomers and complexes via a “hitchhiking” mechanism (5). Overall, these and other secretion types differ in the nature of substrate recognition signal and the mode of substrate translocation.Early secretory antigen 6 kDa (ESX, or type VII) secretion systems are widespread in actinomycetes and Gram-positive bacteria and affect a range of bacterial processes including sporulation, conjugation, and cell wall stability (6–10). In two notorious human pathogens, Mycobacterium tuberculosis and Staphylococcus aureus, ESX secretion was found to be crucial for establishing and maintaining the infection (11–15). Despite the importance of the ESX secretion for human health, the mechanism of this type of secretion is still largely unknown.Recent characterization of the ESX system in Bacillus subtilis confirmed that a functional system is encoded by the yuk/yue operon (16, 17). Importantly, the B. subtilis system codes for a homolog to the prototypical virulence factor substrates from mycobacteria, EsxA and EsxB (18). These proteins all belong to the WXG-100 protein family, which are defined by a conserved WXG amino acid motif that is roughly in the middle of the typically short, otherwise poorly conserved, ∼100-amino-acid polypeptide (Fig. S1A). All characterized WXG proteins share helix-turn-helix hairpin structures where the conserved WXG motif forms a sharp turn between the N- and C-terminal helices (19–23). Two hairpins interact in an antiparallel configuration to form either homo- or heterodimers (Fig. S1B) (19–24). This arrangement places the N and C termini of one subunit in close proximity with the WXG hairpin of the interacting partner. Previous studies have demonstrated that the C-terminal residues and the WXG motif are important for secretion of some mycobacterial substrates but not the others (7, 25, 26). However, dissecting the roles of these motifs has been challenging due to confounding effects, such as their contributions to substrate stability (7, 26, 27). Further complicating the issue, in the mycobacterial system, for example, multiple substrates have been identified and their secretion is codependent (13, 28). There are also multiple closely related ESX systems in mycobacteria that could complicate the analyses (29).Therefore, to investigate the requirements for WXG substrate recognition and the mode of substrate translocation in ESX secretion, we took advantage of the fact that under standard laboratory conditions B. subtilis has a single WXG substrate, YukE (17). Here we report results of a systematic mutagenesis study combined with crosslinking experiments. First, we show that the C-terminal residues of the B. subtilis substrate YukE are important for secretion, suggesting a general mode of recognition of the ESX substrates in firmicutes and actinobacteria. Second, tryptophan and glycine residues of the WXG motif of YukE are required for an efficient transfer of YukE outside of the cell. Third, YukE forms stable homodimers that contain two sites composed by C terminus and WXG turn, but only one intact bipartite site is required for substrate export. Fourth, we present experimental evidence that the ESX system translocates the WXG protein dimers. Together our results show that the ESX system requires a composite signal formed by two folded polypeptides for secretion and exports an intact protein complex that possesses the bipartite signal.