Homologous Toxins Research Articles

A natural point mutation changes both target selectivity and mechanism of action of sea anemone toxins

APETx3, a novel peptide isolated from the sea anemone Anthopleura elegantissima, is a naturally occurring mutant from APETx1, only differing by a Thr to Pro substitution at position 3. APETx1 is believed to be a selective modulator of human ether-á-go-go related gene (hERG) potassium channels with a K(d) of 34 nM. In this study, APETx1, 2, and 3 have been subjected to an electrophysiological screening on a wide range of 24 ion channels expressed in Xenopus laevis oocytes: 10 cloned voltage-gated sodium channels (Na(V) 1.2-Na(V)1.8, the insect channels DmNa(V)1, BgNa(V)1-1a, and the arachnid channel VdNa(V)1) and 14 cloned voltage-gated potassium channels (K(V)1.1-K(V)1.6, K(V)2.1, K(V)3.1, K(V)4.2, K(V)4.3, K(V)7.2, K(V)7.4, hERG, and the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels and provides this toxin the ability to become a potent (EC(50) 276 nM) modulator of voltage-gated sodium channels (Na(V)s) because it slows down the inactivation of mammalian and insect Na(V) channels. Our study also shows that the homologous toxins APETx1 and APETx2 display promiscuous properties since they are also capable of recognizing Na(V) channels with IC(50) values of 31 nM and 114 nM, respectively, causing an inhibition of the sodium conductance without affecting the inactivation. Our results provide new insights in key residues that allow these sea anemone toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins thus far believed to be highly selective.

Crystal Structure of Human ADP-ribose Transferase ARTD15/PARP16 Reveals a Novel Putative Regulatory Domain

ADP-ribosylation is involved in the regulation of DNA repair, transcription, and other processes. The 18 human ADP-ribose transferases with diphtheria toxin homology include ARTD1/PARP1, a cancer drug target. Knowledge of other family members may guide therapeutics development and help evaluate potential drug side effects. Here, we present the crystal structure of human ARTD15/PARP16, a previously uncharacterized enzyme. ARTD15 features an α-helical domain that packs against its transferase domain without making direct contact with the NAD(+)-binding crevice or the donor loop. Thus, this novel domain does not resemble the regulatory domain of ARTD1. ARTD15 displays auto-mono(ADP-ribosylation) activity and is affected by canonical poly(ADP-ribose) polymerase inhibitors. These results add to a framework that will facilitate research on a medically important family of enzymes.

Molecular Determinants of Ligand Selectivity for the Human Multidrug and Toxin Extruder Proteins MATE1 and MATE2-K

The present study compared the selectivity of two homologous transport proteins, multidrug and toxin extruders 1 and 2-K (MATE1 and MATE2-K), and developed three-dimensional pharmacophores for inhibitory ligand interaction with human MATE1 (hMATE1). The human orthologs of MATE1 and MATE2-K were stably expressed in Chinese hamster ovary cells, and transport function was determined by measuring uptake of the prototypic organic cation (OC) substrate 1-methyl-4-phenylpyridinium (MPP). Both MATEs had similar apparent affinities for MPP, with K(tapp) values of 4.4 and 3.7 μM for MATE1 and MATE2-K, respectively. Selectivity was assessed for both transporters from IC(50) values for 59 structurally diverse compounds. Whereas the two transporters discriminated markedly between a few of the test compounds, the IC(50) values for MATE1 and MATE2-K were within a factor of 3 for most of them. For hMATE1 there was little or no correlation between IC(50) values and the individual molecular descriptors LogP, total polar surface area, or pK(a). The IC(50) values were used to generate a common-features pharmacophore, quantitative pharmacophores for hMATE1, and a bayesian model suggesting molecular features favoring and not favoring the interaction of ligands with hMATE1. The models identified hydrophobic regions, hydrogen bond donor and hydrogen bond acceptor sites, and an ionizable (cationic) feature as key determinants for ligand binding to MATE1. In summary, using a combined in vitro and computational approach, MATE1 and MATE2-K were found to have markedly overlapping selectivities for a broad range of cationic compounds, including representatives from seven novel drug classes of Food and Drug Administration-approved drugs.

Snake venomics of Macrovipera mauritanica from Morocco, and assessment of the para-specific immunoreactivity of an experimental monospecific and a commercial antivenoms

Proteomic analysis of the venom of the medically relevant snake Macrovipera mauritanica from Morocco revealed a complex proteome composed of at least 45 toxins from 9 protein families targeting the hemostatic system of the prey or victim. The toxin profile of Moroccan M. mauritanica displays great similarity, but also worth noting departures, with the previously reported venom proteome of M. lebetina from Tunisia. Despite fine compositional differences between these Macrovipera taxa, their overall venom phenotypes explain the clinical picture observed in M. mauritanica and M. lebetina envenomings. However, M. mauritanica venom also contains significant amounts of orphan molecules whose presence in the venom seems to be difficult to rationalize in the context of a predator-prey arms race. The paraspecific immunoreactivity of an experimental monospecific (M. mauritanica) antivenom and a commercial bivalent antivenom, anti-C. cerastes and anti-M. lebetina, against the venoms of Moroccan M. mauritanica and Tunisian M. lebetina, was also investigated through an affinity chromatography-based antivenomics approach. Both antivenoms very efficiently immunodepleted homologous venom toxins and displayed a high degree of paraspecificity, suggesting the clinical utility of the two antivenoms for treating bites of both M. mauritanica or M. lebetina.

Differential expression of the demosponge (Suberites domuncula) carotenoid oxygenases in response to light: protection mechanism against the self-produced toxic protein (Suberitine).

The demosponge Suberites domuncula has been described to contain high levels of a proteinaceous toxin, Suberitine, that displays haemolytic activityIn the present study this 7–8 kDa polypeptide has been isolated and was shown to exhibit also cytotoxic effects on cells of the same species. Addition of retinal, a recently identified metabolite of β-carotene that is abundantly present in S. domuncula was found to reduce both the haemolytic and the cell toxic activity of Suberitine at a molar ratio of 1:1. Spectroscopic analyses revealed that the interaction between β-carotene and Suberitine can be ascribed to a reversible energy transfer reaction. The enzyme that synthesises retinal in the sponge system is the β,β-carotene-15,15′-dioxygenase [carotene dioxygenase]. In order to clarify if this enzyme is the only β-carotene-metabolizing enzyme a further oxygenase had been identified and cloned, the (related) carotenoid oxygenase. In contrast to the dioxygenase, the carotenoid oxygenase could not degrade β-carotene or lycopene in Escherichia coli strains that produced these two carotenoids; therefore it had been termed related-carotenoid oxygenase. Exposure of primmorphs to light of different wavelengths from the visible spectrum resulted after 3 days in a strong upregulation of the dioxygenase in those 3D-cell aggregates that had been incubated with β-carotene. The strongest effect is seen with blue light at a maximum around 490 nm. It is concluded that the toxin Suberitine is non-covalently modified by retinal, the cleavage product from β-carotene via the enzyme carotene dioxygenase, a light inducible oxygenase. Hence, this study highlights that in S. domuncula the bioactive metabolite, retinal, has the property to detoxify its homologous toxin.

A Natural Point Mutation Reveals Target Promiscuity of Toxins Isolated from the Sea Anemone Anthopleura Elegantissima

Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APETx3 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 42 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APETx3 is a natural occurring mutant from APETx1, only differing in 1 amino acid at position 3. APETx1 is believed to be a selective modulator of human ether-a-go-go related (hERG) potassium channels. In this study, APETx1, 2 and 3 have been subjected to an electrophysiological screening on a wide range of 21 ion channels expressed in Xenopus leavis oocytes: 10 cloned voltage-gated sodium channels (NaV1.2-NaV1.8, the insect channels DmNaV1, BgNaV1-1a and the arachnid channel VdNaV1) and 11 cloned voltage-gated potassium channels (KV2.1, KV3.1, KV4.2, KV4.3, KV7.1, KV7.2, KV7.3, KV7.4, KV7.5, hERG, the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels. However, the same substitution provides this toxin the ability to become a potent modulator of voltage-gated sodium channels (NaVs). APETx3 slows down the inactivation of mammalian and insect channels similar to site 3 toxins such as α-scorpion toxins and sea anemone NaVs toxins. Our screening reveals that the homologous toxins APETx1 and APETx2 display promiscuous properties as they are also capable of recognizing NaV channels, causing an inhibition of the sodium conductance.All together, these data provide new insights in key residues which allow these toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins believed to be highly selective.

Preparation and Identification of Monoclonal Antibody against Abrin-a

BALB/c mice were immunized four times with formalin-prepared abrin-a. Using the polyethylene glycol method, immunized splenocytes were isolated and fused with SP2/0 cells. An indirect ELISA was established and used to detect positive clones secreting monoclonal antibodies (mAbs) against abrin-a. After analysis, three hybridoma clones secreting IgG-subtype mAbs were obtained. The antibodies were purified from the hybridoma growth medium using protein A or G affinity chromatography. Western blot analysis was used to analyze the antigenic epitopes on abrin-a recognized by the mAbs. The mAbs were specific for abrin-a, with no detectable cross-reactivity with several homologous toxins and associated agglutinins. Sandwich ELISA was then developed using these mAbs, which had a detection limit for abrin-a of 7.8 ng/mL.

Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

A colloidal gold probe-based silver enhancement immunochromatographic assay for the rapid detection of abrin-a

High-affinity anti-abrin-a monoclonal and polyclonal antibodies were used to develop a sandwich immunochromatographic assay and silver enhancement technology was used to further increase the sensitivity. Using a matrix of double distilled water or soybean milk with added abrin-a, the visual detection limit was found to be 10 ng mL −1. The detection limit was 0.1 ng mL −1 for abrin-a, an increase in sensitivity of 100-fold when the silver enhancement technology was employed. The assay was portable and very simple to perform and the detection was completed within 20 min without complicated handling procedures. There was no significant cross-reactivity with several homologous toxins and associated agglutinin. The assay reagents could be stored for 12 weeks at 4 °C without significant loss of activity. These characteristics make the strip assay to be an ideal candidate for the development of a rapid toxin detection kit.

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure-function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC(50) of 1.70 n± 1.35 µM, whereas 10 µM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 µM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca(2+) channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K(+) channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.

Listeria monocytogenesInduces T Cell Receptor Unresponsiveness through Pore‐Forming Toxin Listeriolysin O

The success of many pathogens relies on their ability to circumvent the innate and adaptive immune defenses. How bacterial pathogens subvert adaptive immune defenses is not clear. Cholesterol-dependent cytolysins (CDCs) represent an expansive family of homologous pore-forming toxins that are produced by more than 20 gram-positive bacterial species. Listeriolysin O (LLO), a prototype CDC, is the main virulence factor of Listeria monocytogenes. We employed flow cytometric and microarray techniques to analyze the effect of LLO on T cell activation in vitro and in vivo. In vivo and in vitro proliferation of CD4(+) T cells upon T cell receptor (TCR) activation was highly diminished in the presence of LLO or wild-type L. monocytogenes but not in the presence of LLO-deficient L. monocytogenes. This block in T cell proliferation was specific to T cell activation via the TCR and not by phorbol 12-myristate 13-acetate-ionomycin, which bypasses the proximal TCR signaling event. The results of microarray analysis suggest that LLO-induced T cell unresponsiveness is due to the induction of a calcium-nuclear factor of activated T cells-dependent transcriptional program that drives the expression of negative regulators of TCR signaling. These findings provide important insights into how bacterial toxins silence adaptive immune responses and thus enable prolonged survival of the pathogen in the host.

Multidrug And Toxin Extrusion's (MATE) Role in Renal Organic Cation Secretion

The molecular identity of the luminal element of renal organic cation (OC) secretion is suspected to include two novel homologous transport proteins, Multidrug And Toxin Extruders 1 & 2K (MATE1 and MATE2K); they share anticipated characteristics of luminal OC transport, including expression in the apical membrane of human renal proximal tubule (RPT) cells and support of OC/H+ exchange. Here we investigated aspects of the multispecificity of human MATE1 (hMATE1) and MATE2K (hMATE2K). Stable transfection in Chinese Hamster Ovary cells resulted in Kt values for transport of 1‐methyl‐4‐phenylpyridinium (MPP) of 7.6 μM (MATE1) 5.2 μM (MATE2K). Inhibition by tetraethylammonium produced IC50 values of 52 μM and 26 μM for hMATE1 and hMATE2K, respectively. Preliminary data shows that cimetidine is a potent inhibitor of MPP, with IC50s of 1 μM (MATE1) and 3μM (MATE2K). Increasing extracellular H+ concentration cis inhibited hMATE1‐mediated MPP transport with an IC50 value of 15.4 nM (pH of 7.8). Immunohistochemical analysis of paraffin embedded human kidney cortex samples confirmed that hMATE1 and hMATE2K are located in the apical membrane of RPT cells and initial evidence suggests a heterogeneous distribution of both transporters in the cortical RPTs. In conclusion, the selectivities and distribution of MATE1 and MATE2K support the contention that they play a key role in OC secretion in human RPT. (F31DK084769)

Complete genomic sequence of bacteriophage felix o1.

Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage φEa21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent.

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 A x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a beta-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a pi-cation interaction within the beta-flap that appear to be unique among the large clostridial cytotoxins.

Hamiltonella defensa , genome evolution of protective bacterial endosymbiont from pathogenic ancestors

Eukaryotes engage in a multitude of beneficial and deleterious interactions with bacteria. Hamiltonella defensa, an endosymbiont of aphids and other sap-feeding insects, protects its aphid host from attack by parasitoid wasps. Thus H. defensa is only conditionally beneficial to hosts, unlike ancient nutritional symbionts, such as Buchnera, that are obligate. Similar to pathogenic bacteria, H. defensa is able to invade naive hosts and circumvent host immune responses. We have sequenced the genome of H. defensa to identify possible mechanisms that underlie its persistence in healthy aphids and protection from parasitoids. The 2.1-Mb genome has undergone significant reduction in size relative to its closest free-living relatives, which include Yersinia and Serratia species (4.6-5.4 Mb). Auxotrophic for 8 of the 10 essential amino acids, H. defensa is reliant upon the essential amino acids produced by Buchnera. Despite these losses, the H. defensa genome retains more genes and pathways for a variety of cell structures and processes than do obligate symbionts, such as Buchnera. Furthermore, putative pathogenicity loci, encoding type-3 secretion systems, and toxin homologs, which are absent in obligate symbionts, are abundant in the H. defensa genome, as are regulatory genes that likely control the timing of their expression. The genome is also littered with mobile DNA, including phage-derived genes, plasmids, and insertion-sequence elements, highlighting its dynamic nature and the continued role horizontal gene transfer plays in shaping it.

Production of catalytically inactive BoNT/A1 holoprotein and comparison with BoNT/A1 subunit vaccines against toxin subtypes A1, A2, and A3

A recombinant, catalytically inactive Clostridium botulinum neurotoxin A1 holoprotein (ciBoNT/A1 HP) was constructed by introducing amino acid substitutions H223A, E224A, and H227A in the active site to ablate proteolytic activity. ciBoNT/A1 HP was produced in the yeast Pichia pastoris and the purified product was evaluated as a vaccine candidate by comparison against recombinant BoNT/A1 LC, LC-belt, LC-H n, and H c antigens and a LC-H n + H c combination in mouse potency and efficacy bioassays when challenged with BoNT/A subtypes /A1, /A2, and /A3. A single dose of ciBoNT/A1 HP provided equivalent or greater protective immunity, not only against the homologous toxin, but also against two distinct toxin subtypes with significant amino acid divergence. Only the LC-H n + H c combination provided comparable protection against /A1; however, it was less effective against subtypes /A2 and /A3. Differences in protective immunity diminished after multiple vaccinations with either ciBoNT/A1 HP or BoNT/A1 H c, and the survival rates were more comparable at the toxin levels used to challenge.

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV1000) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV100) were more appropriate for discriminating an all-or-none leakage (α=0) from the graded leakage with low values of α. While disrupting LUV1000, CyaA caused a highly cation-selective leakage (α~15) whereas its mutated form with decreased channel K+/Cl− selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K+E516K) exhibited much lower selectivity (α∼6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.

Killing of Rat Basophilic Leukemia Cells by Lethal Toxin from Clostridium sordellii: Critical Role of Phosphatidylinositide 3′-OH Kinase/Akt Signaling

Clostridium sordellii lethal toxin (TcsL) belongs to the family of clostridial glucosylating toxins. TcsL exhibits glucosyltransferase activity to inactivate Rho and Ras proteins. On cultured cells, TcsL causes actin reorganization ("cytopathic effect") and apoptotic cell death ("cytotoxic effect"). This study is based on the concept that the cytotoxic effects of TcsL depend on the glucosylation of critical substrate proteins rather than on the glucosyltransferase activity per se. The cytotoxic effects of TcsL depend on the glucosyltransferase activity of TcsL, as neither chemically inactivated TcsL nor a glucosyltransferase-deficient mutant version of TcsL caused it. The TcsL homologous toxin B from Clostridium difficile serotype F strain 1470 (TcdBF) also failed to cause cytotoxic effects. Correlation of the toxins' respective protein substrate specificities highlighted (H/K/N)Ras as critical substrate proteins for the cytotoxic effects. (H/K/N)Ras are critical upstream regulators of phosphatidylinositide 3'-OH kinase (PI3K)/Akt survival signaling. Tauroursodeoxycholic acid (TUDCA) classified to activate PI3K/Akt signaling downstream of apoptosis-inducing stimuli prevented the cytotoxic effects of TcsL. In conclusion, (H/K/N)Ras glucosylation and subsequent inhibition of PI3K/Akt signaling are critical for the cytotoxic effects of TcsL.

Protein-Protein Docking and Analysis Reveal That Two Homologous Bacterial Adenylyl Cyclase Toxins Interact with Calmodulin Differently

Calmodulin (CaM), a eukaryotic calcium sensor that regulates diverse biological activities, consists of N- and C-terminal globular domains (N-CaM and C-CaM, respectively). CaM serves as the activator of CyaA, a 188-kDa adenylyl cyclase toxin secreted by Bordetella pertussis, which is the etiologic agent for whooping cough. Upon insertion of the N-terminal adenylyl cyclase domain (ACD) of CyaA to its targeted eukaryotic cells, CaM binds to this domain tightly ( approximately 200 pm affinity). This interaction activates the adenylyl cyclase activity of CyaA, leading to a rise in intracellular cAMP levels to disrupt normal cellular signaling. We recently solved the structure of CyaA-ACD in complex with C-CaM to elucidate the mechanism of catalytic activation. However, the structure of the interface between N-CaM and CyaA, the formation of which contributes a 400-fold increase of binding affinity between CyaA and CaM, remains elusive. Here, we used site-directed mutations and molecular dynamic simulations to generate several working models of CaM-bound CyaA-ACD. The validity of these models was evaluated by disulfide bond cross-linking, point mutations, and fluorescence resonance energy transfer experiments. Our study reveals that a beta-hairpin region (amino acids 259-273) of CyaA-ACD likely makes contacts with the second calcium binding motif of the extended CaM. This mode of interaction differs from the interaction of N-CaM with anthrax edema factor, which binds N-CaM via its helical domain. Thus, two structurally conserved, bacterial adenylyl cyclase toxins have evolved to utilize distinct binding surfaces and modes of activation in their interaction with CaM, a highly conserved eukaryotic signaling protein.

From soil to gut:Bacillus cereusand its food poisoning toxins

Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrhoeal type of food-associated illness that is becoming increasingly important in the industrialized world. The emetic disease is a food intoxication caused by cereulide, a small ring-formed dodecadepsipeptide. Similar to the virulence determinants that distinguish Bacillus thuringiensis and Bacillus anthracis from B. cereus, the genetic determinants of cereulide are plasmid-borne. The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores, thought to produce protein enterotoxins in the small intestine. Three pore-forming cytotoxins have been associated with diarrhoeal disease: haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K. Hbl and Nhe are homologous three-component toxins, which appear to be related to the monooligomeric toxin cytolysin A found in Escherichia coli. This review will focus on the toxins associated with foodborne diseases frequently caused by B. cereus. The disease characteristics are described, and recent findings regarding the associated toxins are discussed, as well as the present knowledge on virulence regulation.