Domain Of Toxin Research Articles

New players in the toxin field: polymorphic toxin systems in bacteria.

ABSTRACTBacteria have evolved numerous strategies to increase their competitiveness and fight against each other. Indeed, a large arsenal of antibacterial weapons is available in order to inhibit the proliferation of competitor cells. Polymorphic toxin systems (PTS), recently identified by bioinformatics in all major bacterial lineages, correspond to such a system primarily involved in conflict between related bacterial strains. They are typically composed of a secreted multidomain toxin, a protective immunity protein, and multiple cassettes encoding alternative toxic domains. The C-terminal domains of polymorphic toxins carry the toxic activity, whereas the N-terminal domains are related to the trafficking mode. In silico analysis of PTS identified over 150 distinct toxin domains, including putative nuclease, deaminase, or peptidase domains. Immunity genes found immediately downstream of the toxin genes encode small proteins that protect bacteria against their own toxins or against toxins secreted by neighboring cells. PTS encompass well-known colicins and pyocins, contact-dependent growth inhibition systems which include CdiA and Rhs toxins and some effectors of type VI secretion systems. We have recently characterized the MafB toxins, a new family of PTS deployed by pathogenic Neisseria spp. Many other putative PTS have been identified by in silico predictions but have yet to be characterized experimentally. However, the high number of these systems suggests that PTS have a fundamental role in bacterial biology that is likely to extend beyond interbacterial competition.

Intraspecies Competition in Serratia marcescens Is Mediated by Type VI-Secreted Rhs Effectors and a Conserved Effector-Associated Accessory Protein.

ABSTRACTThe type VI secretion system (T6SS) is widespread in Gram-negative bacteria and can deliver toxic effector proteins into eukaryotic cells or competitor bacteria. Antibacterial T6SSs are increasingly recognized as key mediators of interbacterial competition and may contribute to the outcome of many polymicrobial infections. Multiple antibacterial effectors can be delivered by these systems, with diverse activities against target cells and distinct modes of secretion. Polymorphic toxins containing Rhs repeat domains represent a recently identified and as-yet poorly characterized class of T6SS-dependent effectors. Previous work had revealed that the potent antibacterial T6SS of the opportunistic pathogen Serratia marcescens promotes intraspecies as well as interspecies competition (S. L. Murdoch, K. Trunk, G. English, M. J. Fritsch, E. Pourkarimi, and S. J. Coulthurst, J Bacteriol 193:6057–6069, 2011, http://dx.doi.org/10.1128/JB.05671-11). In this study, two new Rhs family antibacterial effectors delivered by this T6SS have been identified. One of these was shown to act as a DNase toxin, while the other contains a novel, cytoplasmic-acting toxin domain. Importantly, using S. marcescens, it has been demonstrated for the first time that Rhs proteins, rather than other T6SS-secreted effectors, can be the primary determinant of intraspecies competition. Furthermore, a new family of accessory proteins associated with T6SS effectors has been identified, exemplified by S. marcescens EagR1, which is specifically required for deployment of its associated Rhs effector. Together, these findings provide new insight into how bacteria can use the T6SS to deploy Rhs-family effectors and mediate different types of interbacterial interactions. IMPORTANCE Infectious diseases caused by bacterial pathogens represent a continuing threat to health and economic prosperity. To counter this threat, we must understand how such organisms survive and prosper. The type VI secretion system is a weapon that many pathogens deploy to compete against rival bacterial cells by injecting multiple antibacterial toxins into them. The ability to compete is vital considering that bacteria generally live in mixed communities. We aimed to identify new toxins and understand their deployment and role in interbacterial competition. We describe two new type VI secretion system-delivered toxins of the Rhs class, demonstrate that this class can play a primary role in competition between closely related bacteria, and identify a new accessory factor needed for their delivery.

A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model.

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and B in vitro and because of their in vivo efficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains of C. difficile toxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

Genetic analysis of the CDI pathway from Burkholderia pseudomallei 1026b.

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIII Bp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIII Bp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIII Bp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTII Bp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI+ inhibitor cells to the same extent as BTH_I0986+ cells, suggesting that LPS could function as a receptor for CdiAII Bp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIII Bp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species.

Intracellular plasma membrane guidance of Photorhabdus asymbiotica toxin is crucial for cell toxicity.

The bacterial toxin Photorhabdus asymbiotica toxin (PaTox) modifies Rho proteins by tyrosine GlcNAcylation and heterotrimeric Gα proteins by deamidation. Inactivation of Rho proteins results in F-actin disassembly in host cells. Here, we analyzed the subcellular distribution of PaTox and show that the glycosyltransferase domain of PaTox associates with the negatively charged inner surface of the plasma membrane. Localization studies with site-directed mutants, liposome precipitation analysis, lipid overlay assays, and confocal time-lapse microscopy revealed that a patch of positively charged lysine and arginine residues located on helix α1 of the glycosyltransferase is essential for membrane attachment. Using a helix1 deletion mutant, we show that plasma membrane localization of PaTox is essential for cytotoxicity and proved this by substitution of helix1 by an N-terminal myristoylation signal peptide, which restored plasma membrane localization and cytotoxicity. Furthermore, we also show that the intracellular deamidase activity of PaTox depends on the presence of the membrane localization domain. Comparison of PaTox membrane-binding domain with the 4-helix-bundle membrane-binding domain of Pasteurella multocida toxin, Vibrio cholerae multifunctional autoprocessing repeats-in-toxin, and clostridial glucosylating toxins revealed similar spatial geometry and charge distribution but different structural topology, indicating convergent evolution of toxin domains for optimized host target interaction.

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin.

Functional assembly of 260-kDa oligomers required for mosquito-larvicidal activity of the Bacillus thuringiensis Cry4Ba toxin

Oligomerization has been shown to contribute to the toxicity of Bacillus thuringiensis Cry toxins. Mutations have been made in the Cry4Ba toxin and resulted in toxic to non-toxic mutants toward Aedes aegypti larvae. In this study, Cry4Ba wild type and mutants were analyzed for oligomer formation in vitro, biochemical properties and their relationships with larvicidal activity. In vitro, the Cry4Ba forms two-main types of the oligomers including (1) the 260-kDa and larger oligomers, which assembled in the carbonate buffer, pH 10.0 and completely dissociated by heating at 90°C and (2) 190-kDa oligomer, which was induced by heat, sodium-salt and detergent addition. Polar and charge residues in the toxin domain I and II may contribute to formation of the 260-kDa oligomers. A single Cys-525 in domain III was replaced with serine resulting in the C525S mutant, which exhibited a 50% reduction in larvicidal activity compared to the Cry4Ba wild-type. The mutant exhibited partial loss in larger oligomer of the 260kDa and total loss of 190-kDa oligomer. The results revealed an important role of the Cys-525 in intermolecular disulfide formation of larger oligomer as well as the 190-kDa oligomer. Despite of their formations in the receptor free condition, the 260-kDa and larger oligomers were found to strongly correspond to Cry4Ba toxicity suggesting their functional roles in the A. aegypti larvae. Also, possible roles of the 260-kDa and larger oligomers have been proposed in this report.

Functions of cholera toxin B-subunit as a raft cross-linker.

Lipid rafts are putative complexes of lipids and proteins in cellular membranes that are proposed to function in trafficking and signalling events. CTxB (cholera toxin B-subunit) has emerged as one of the most studied examples of a raft-associated protein. Consisting of the membrane-binding domain of cholera toxin, CTxB binds up to five copies of its lipid receptor on the plasma membrane of the host cell. This multivalency of binding gives the toxin the ability to reorganize underlying membrane structure by cross-linking otherwise small and transient lipid rafts. CTxB thus serves as a useful model for understanding the properties and functions of protein-stabilized domains. In the present chapter, we summarize current evidence that CTxB associates with and cross-links lipid rafts, discuss how CTxB binding modulates the architecture and dynamics of membrane domains, and describe the functional consequences of this cross-linking behaviour on toxin uptake into cells via endocytosis.

Identification and characterization of Clostridium perfringens beta toxin variants with differing trypsin sensitivity and in vitro cytotoxicity activity.

By producing toxins, Clostridium perfringens causes devastating diseases of both humans and animals. C. perfringens beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin.

Identification and validation of a linear protective neutralizing epitope in the β-pore domain of alpha toxin.

The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha toxin could facilitate the development of an epitope-focused vaccine against S. aureus.

Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel.

Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 α helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1-TH4, translocates across the membrane to the trans side. Residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6-TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6-TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue's accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6-TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix.

Biochemical and Immunological Characterizations of the Receptor Binding Domain of C. difficile Toxin B

Clostridium difficile (Cd) is an emerging nosocomial pathogen responsible for antibiotic-associated pseudo-membranous colitis and diarrhea in hospital acquired infections. Clostridial toxins A (TcdA) and B (TcdB) which specifically bind to unknown glycoprotein(s) on the surface of epithelial cells disrupt the intestinal barrier and ultimately lead to acute inflammation and diarrhea . There is still debate as to whether the receptor binding domains (RBD) of toxins can individually elicit protection in the hamster challenge model. In this study, a TcdB RBD which was derived from C.difficile strain VPI10463 with >95% amino acid sequence identity to hyper-virulent strain BI/NAP1/027 was designed and expressed in Escherichia coli. Recombinant RBD (rRBD) was purified, characterized biologically and immunologically and found to have the following properties: (a) capable of binding to the cell surface of both Vero and Caco-2 cells and entering into the cytosol; (b) devoid of hemagglutinin activity (HA); (c) the ability to up-regulate cell surface markers expressions and cytokines secretions from dendritic cells; (d) eliciting anti-TcdB neutralizing antibody responses that could weakly cross-neutralize TcdA in the absence of adjuvant; (e) and inducing weak protection against a lethal dose of Cd spores in the hamster challenge model. Therefore, rRBD shows potential as an immunogen to be included in the development of vaccines against Clostridium difficile-associated diseases.

Ribosome Inactivating Proteins: Exploiting Plant Weapons to Fight Human Cancer

Ribosomeinactivating proteins (RIPs) are potent toxins from plant or bacterial origin that work by irreversibly block ribosomes of target cells, causing apoptotic cell death. Their noteworthy activity has been exploited to therapeutic purpose in the treatment of cancer. The specificity for malignant cells has been addressed by using targeting domains such as antibodies to form immunotoxins or ligands to get chimeric fusions. These therapeutics have shown encouraging preliminary results in hematological tumors as well as in solid tumors with the most effective ones currently undergoing clinical trials. Denileukin diftitox, a fusion between the catalytic domain of the diphtheria toxin and human cytokine interleukin-2, was the first toxin-derived drug approved by the FDA. Another, more recent way to take advantage of RIPs is to utilize their DNA in the suicide gene therapy. Toxin DNA can be delivered trough a vector to or complexed and directly injected in the tumor. In the future RIPs are expected in to give a significant contribution in the treatment of the cancer, also in combination with traditional therapies. The scope of this review is to highlight advantages and drawbacks of RIP based biomedical applications, by evaluating their employment as either targeted proteins or genes as effectors.

Design and evaluation of a peptide-based immunotoxin for breast cancer therapeutics

Immunotoxins are chimeric proteins comprising a specific cellular targeting domain linked to a cytotoxic factor. Here we describe the design and use of a novel, peptide-based immunotoxin that can initiate selective cytotoxicity on ErbB2-positive cells. ErbB2 is a receptor tyrosine kinase that is overexpressed in the tumor cells of approximately 30% of breast cancer patients. Immunotoxin candidates were designed to incorporate a targeting ligand with affinity for ErbB2 along with a membrane lysin-based toxin domain. One particular peptide candidate, NL1.1-PSA, demonstrated selective cytotoxicity towards ErbB2-overexpressing cell lines. We utilized a bioengineering strategy to show that recombinant NL1.1-PSA immunotoxin expression by Escherichia coli also conferred selective cytotoxicity towards ErbB2-overexpressing cells. Our findings hold significant promise for the use of effective immunotoxins in cancer therapeutics.

Remission Induction in a Phase 1 Study of a Bispecific Single Chain Immunotoxin Targeting CD22 and CD19 (DT2219) for Refractory B-Cell Malignancies

DT2219 is a recombinant fusion protein composed of catalytic and translocation domains of diphtheria toxin (DT) fused with bispecific single chain variable fragments (sFv) of antibodies targeting human CD19 and CD22 cell surface receptors. Preclinical testing determined that the combination of two different sFvs and a toxin on the same single-chain molecule resulted in efficient toxin delivery and greater anticancer activity compared to monomeric immunotoxins sFv alone or anti-CD19 and anti-CD22 parental monoclonal antibodies. Murine studies demonstrated clinical efficacy and enhanced therapeutic effect with repetitive dosing (Vallera D, Clin Cancer Res 2009).We conducted a phase 1 study to assess safety, to determine the maximum tolerated dose (MTD) and to evaluate preliminary efficacy in patients with chemotherapy refractory B cell leukemia or lymphoma expressing CD19 and/or CD22. DT2219 was administered in a single course intravenously over 4 hours (1-2 hours for subsequent doses) every other day for 4 total doses. The starting dose was 0.5 µg/kg/dose (2.0 ug/kg/course) (1/1000th of the MTD in rabbits). The dose was escalated by accelerated design until dose limited toxicity (DLT) was observed. Twenty-two patients were enrolled, with a median age of 55 years (range, 34-78). Five patients had pre-B acute lymphoblastic leukemia (4 refractory, 1 with extra-medullary disease, 1 had chronic lymphocytic leukemia (CLL) and 7 had non-Hodgkin lymphoma). All were chemo-refractory with a median of 4 prior therapies. All patients had biopsy-confirmed tumor expression of CD19 and/or CD22. The first 9 patients were treated at doses ranging from 0.5 ug/kg/dose to 20 ug/kg/dose. No drug was detectable in those patients and they experienced no DLT or clinical responses. DT2219 was detectable in the serum at doses ≥40 µg/kg/dose (N=13). Dose levels were 40 µg/kg/day N=5; 60 µg/kg/day N=5; 80 µg/kg/day N=3. The area under the curve (AUC ) range was 1,104 -1,346 and the half-life ranged from 54-84 minutes. After a single course of DT2219 we observed partial remission in a 77 year old patient with rituximab/chemotherapy-refractory CLL (dose level 40 µg/kg). The 40% reduction in the abdominal tumor mass is shown in Figure 1. This patient did not experience any DLT. A second partial remission was observed in 53 year old patient with relapsed marginal zone lymphoma (dose level 60 µg/kg) who experienced a DLT of capillary leak syndrome. After regulatory approval, that patient received a second treatment course 8 weeks later at a reduced dose of 40 µg/kg which resulted in a complete remission (Figure 2). Both patients are alive and in remission at 6 and 4 months, respectively, after therapy. Adverse events were observed in all patients who received ≥40 µg/kg. The most common was grade 1-2 capillary leak syndrome, grade 1-2 hematologic toxicity, elevated liver function tests and fatigue. Two patients experienced DLTs: at the 40 µg/kg dose (grade 3 lower extremity weakness) and 60 µg/kg dose ( grade 4 neutropenia and grade 3 capillary leak). All adverse reactions resolved completely within one week. Immunogenicity and formation of neutralizing antibodies is a major barrier in development of DT-based immunotoxins. Remarkably, none of the 8 patients with B-cell lymphoma/CLL who had received rituximab within weeks preceding therapy with DT2219 had detectable neutralizing antibody. These patients had no detectable B cells at the time of study drug administration. This suggests that rituximab pre-treatment may prevent the development of neutralizing antibodies and allow for repetitive dosing. In conclusion, we have demonstrated promising clinical efficacy of the novel immunotoxin DT2219 in refractory B cell lymphoid malignancies. We determined that the biologically active dose of DT2219 is between 40-80 µg/kg and that repetitive dosing is feasible in patients pre-treated with rituximab. Phase 2 trial is in development. [Display omitted] [Display omitted] DisclosuresVallera:The Lion Fund, William Lawrence and Blanche Hughes Foundation : Other, Research Funding.

Hydrogen-deuterium exchange and mass spectrometry reveal the pH-dependent conformational changes of diphtheria toxin T domain.

The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen–deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our “standard condition” (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W+-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8–9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8–9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain.

Comparative Analysis of the Secretome from a Model Filarial Nematode (Litomosoides sigmodontis) Reveals Maximal Diversity in Gravid Female Parasites

Filarial nematodes (superfamily Filarioidea) are responsible for an annual global health burden of ∼6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans. No vaccine exists for the major filarial diseases, lymphatic filariasis and onchocerciasis; in part because research on protective immunity against filariae has been constrained by the inability of the human-parasitic species to complete their lifecycles in laboratory mice. However, the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction. Here, we provide a comprehensive analysis of excretory-secretory products from L. sigmodontis across five lifecycle stages and identifications of host proteins associated with first-stage larvae (microfilariae) in the blood. Applying intensity-based quantification, we determined the abundance of 302 unique excretory-secretory proteins, of which 64.6% were present in quantifiable amounts only from gravid adult female nematodes. This lifecycle stage, together with immature microfilariae, released four proteins that have not previously been evaluated as vaccine candidates: a predicted 28.5 kDa filaria-specific protein, a zonadhesin and SCO-spondin-like protein, a vitellogenin, and a protein containing six metridin-like ShK toxin domains. Female nematodes also released two proteins derived from the obligate Wolbachia symbiont. Notably, excretory-secretory products from all parasite stages contained several uncharacterized members of the transthyretin-like protein family. Furthermore, biotin labeling revealed that redox proteins and enzymes involved in purinergic signaling were enriched on the adult nematode cuticle. Comparison of the L. sigmodontis adult secretome with that of the human-infective filarial nematode Brugia malayi (reported previously in three independent published studies) identified differences that suggest a considerable underlying diversity of potential immunomodulators. The molecules identified in L. sigmodontis excretory-secretory products show promise not only for vaccination against filarial infections, but for the amelioration of allergy and autoimmune diseases.

The proton-motive force is required for translocation of CDI toxins across the inner membrane of target bacteria.

Contact-dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two-partner secretion proteins. The CdiA effector extends from the surface of CDI(+) inhibitor cells, binds to receptors on neighbouring bacteria and delivers a toxin domain derived from its C-terminal region (CdiA-CT). Here, we show that CdiA-CT toxin translocation requires the proton-motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA-CT translocation is clearly distinct from known colicin-import pathways because ΔtolA ΔtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA-CT toxins can be transferred into the periplasm of de-energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de-energized conditions remain competent to enter the target-cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer- and inner-membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target-cell cytoplasm.

Vibrio Type III Effector VPA1380 Is Related to the Cysteine Protease Domain of Large Bacterial Toxins

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2), but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator.

The VgrG Proteins Are “à la Carte” Delivery Systems for Bacterial Type VI Effectors

The bacterial type VI secretion system (T6SS) is a supra-molecular complex akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas aeruginosa H1-T6SS has been extensively characterized. It is involved in bacterial killing and in the delivery of three toxins, Tse1-3. Here, we demonstrate the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. In this respect, VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C terminus carries a toxic activity, from which the producing bacterium is protected by a cognate immunity. Similarly, VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Overall, these data develop the concept that VgrGs are indispensable components for the specific delivery of effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes. A closer inspection of these clusters reveals that they also encode putative toxins. Overall, these associations further support the notion of an original form of secretion system, in which VgrG acts as the carrier.