Showing papers in "The Open Toxinology Journal in 2013"

Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases.

Abstract: Clostridium perfringens is a gram-positive anaerobic rod that is classified into 5 toxinotypes (A, B, C, D, and E) according to the production of 4 major toxins, namely alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX). However, this microorganism can produce up to 16 toxins in various combinations, including lethal toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). Most diseases caused by this microorganism are mediated by one or more of these toxins. The role of CPA in intestinal disease of mammals is controversial and poorly documented, but there is no doubt that this toxin is essential in the production of gas gangrene of humans and several animal species. CPB produced by C. perfringens types B and C is responsible for necrotizing enteritis and enterotoxemia mainly in neonatal individuals of several animal species. ETX produced by C. perfringens type D is responsible for clinical signs and lesions of enterotoxemia, a predominantly neurological disease of sheep and goats. The role of ITX in disease of animals is poorly understood, although it is usually assumed that the pathogenesis of intestinal diseases produced by C. perfringens type E is mediated by this toxin. CPB2, a necrotizing and lethal toxin that can be produced by all types of C. perfringens, has been blamed for disease in many animal species, but little information is currently available to sustain or rule out this claim. CPE is an important virulence factor for C. perfringens type A gastrointestinal disease in humans and dogs; however, the data implicating CPE in other animal diseases remains ambiguous. PFO does not seem to play a direct role as the main virulence factor for animal diseases, but it may have a synergistic role with CPA-mediated gangrene and ETX-mediated enterotoxemia. The recent improvement of animal models for C. perfringens infection and the use of toxin gene knock-out mutants have demonstrated the specific pathogenic role of several toxins of C. perfringens in animal disease. These research tools are helping us to establish the role of each C. perfringens toxin in animal disease, to investigate the in vivo mechanism of action of these toxins, and to develop more effective vaccines against diseases produced by these microorganisms.

Insecticidal Toxins from the Photorhabdus and Xenorhabdus Bacteria

Abstract: Insect pathogens are an excellent source of novel insecticidal agents with proven toxicity. In particular, bacteria from the genera Photorhabdus and Xenorhabdus are proving to be a genomic goldmine, encoding a multitude of insecticidal toxins. Some are highly specific in their target species, whilst others are more generalist, but all are of potential use in crop protection against insect pests. These astounding bacterial species are also turning out to be equipped to produce a vast range of anti-microbial compounds which could be of use to medical science. This review will cover the current knowledge of the lifecycles of the two genera and the potential role of the toxins in their biology, before a more in depth exploration of some of the best studied toxins and their potential use in agriculture.

Overview of the Basic Biology of Bacillus thuringiensis with Emphasis on Genetic Engineering of Bacterial Larvicides for Mosquito Control

Abstract: The insecticidal bacterium, Bacillus thuringiensis, consists of a wide variety of subspecies, most of which are insecticidal for either lepidopteran, coleopteran, or dipteran insect larvae. Subspecies such as B. thuringiensis subsp. kurstaki have been used with remarkable safety for more than forty years to control lepidopteran pests in agriculture and forestry, and over the past thirty years, B. thruingeinsis subsp. israelensis, has proven to be a safe and effective larvicide for controlling mosquito and black fly larvae. Studies of the basic biology of B. thuringiensis have shown that it produces a variety of insecticidal proteins produced during vegetative growth and sporulation that determines its activity for insect species belonging to different orders, with the most important of these being the Cry proteins active against lepidopteran and coleopteran pests, and a combination of Cry and Cyt proteins for mosquitoes and blackflies. After intoxication by these proteins, spores typically germinate and invade larvae, contributing to insect mortality. Whereas strains of many wild type isolates have been commercialized and are now used worldwide, the use of recombinant DNA techniques, i.e., genetic engineering, has been used over the past decade to recombine the proteins of different B. thuringiensis strains with those of B. sphaericus to generate recombinant larvicides as much as ten-fold more toxic than the parental strains. In this chapter, we begin with a general overview of the basic biology of B. thuringiensis and B. sphaericus, then show how studies of its molecular genetics combined with recombinant DNA techniques have been used to generate highly improved bacterial larvicides for control of nuisance and vector mosquitoes.

Mosquito Resistance to Bacterial Larvicidal Toxins

Abstract: Insecticide resistance to the microbial insecticides Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) represents a serious threat to their success. Available evidence indicates that the risk for resistance to Bti is low due to the makeup of its parasporal crystal, which contains Cyt1A, Cry4A, Cry4B, and Cry11A toxic proteins. Disrupting the toxin complex in Bti enables resistance to evolve, especially in the absence of the key factor, the cytolytic toxin, Cyt1A. Cross-resistance is widespread among mosquitocidal Bacillus thuringiensis Cry toxins and the mechanisms of Cry resistance in mosquitoes are not known. Bacillus sphaericus (Bs) is at higher risk for resistance due to its single- site action and field cases have been reported from a number of locations worldwide. Cross-resistance is reported among the various Bs isolates, although some isolates produce additional toxic proteins that can reduce cross-resistance and slow resistance evolution. Field and lab evolved resistant populations consistently show recessive and monofactorial inheritance of resistance. Resistant populations, however, have evolved a variety of molecular mechanisms causing that resistance. Traditional resistance management strategies with promise include rotations and mixtures of Bti and Bs, as well as untreated areas that provide natural refuges for susceptible alleles. Promising new strategies include genetic engineering to increase the toxin complexity targeted toward mosquito larvae, to enhance the host range of the mosquito control product, and to avoid the evolution of insecticide resistance. Regardless of the control strategy, a resistance- monitoring program alongside an integrative pest management approach is the best strategy to delay insecticide resistance.

The Aerolysin-Like Toxin Family of Cytolytic, Pore-Forming Toxins

Abstract: Pore-forming toxins (PFTs) represent the largest known group of bacterial protein toxins to date. Membrane insertion and subsequent pore-formation occurs after initial binding to cell-surface receptor and oligomerization. Aerolysin, a toxin produced by the Gram-negative bacterium Aeromonas hydrophila and related species, belongs to the PFT group and shares a common mechanism of action involving  -barrel structures resulting from the assembly of  - hairpins from individual toxin monomers into a heptamer. Aerolysin is also the name given to structurally and mechanistically related toxins called the aerolysin-like toxin family. A universal characteristic of this toxin family involves the diverse life forms that synthesize these proteins throughout Nature. Examples include: 1) epsilon-toxin and septicum-alpha-toxin produced by anaerobic, Gram-positive Clostridium species; 2) enterolobin by the Brazilian tree Enterolobium contortisiliquum; 3) a mushroom toxin Laetiporus sulphureus lectin (LSL); 4) mosquitocidal toxins (Mtxs) from the Gram-positive bacteria Bacillus sphaericus and parasporine-2 from Bacillus thuringiensis; and 6) hydralysins from the tiny aquatic animal Chlorohydra viridis. The following review provides an overview of the different members within the aerolysin-like toxin family.

Fate and Effects in Soil of Cry Proteins from Bacillus thuringiensis: Influence of Physicochemical and Biological Characteristics of Soil

Abstract: Bacillus thuringiensis (Bt) is a useful alternative or supplement to synthetic chemical pesticides in agriculture, forest management, and control of mosquitoes and some other biting insects. When modified Bt cry genes are inserted into a plant species (e.g., corn, cotton, potato, canola, rice), the plant expresses active larvicidal proteins in its tissues. The toxins continue to be synthesized during growth of the plants, making the plant toxic to various insect pests throughout their life or as biomass incorporated into soil. If production exceeds consumption, inactivation, and degradation, the toxins could accumulate to concentrations that may enhance the control of target pests or constitute a hazard to nontarget organisms, such as the soil microbiota, beneficial insects (e.g., pollinators, predators and parasites of insect pests), and other animal classes. The accumulation and persistence of the toxins could also result in the selection and enrichment of toxin-resistant target insects. Persistence is enhanced when the toxins are bound on surface-active particles in the environment (e.g., clays and humic substances) and, thereby, rendered more resistant to biodegradation while retaining toxic activity. Moreover, major problem we face today is of "Molecular pharming" that utilizes transgenic plants and animals for production of pharmaceuticals and chemicals for their use in human beings and industries respectively. Their release to the environment, especially to soil and potentially to waters of the pharmaceutical and industrial products of transgenic plant and animal "pharms" could pose a hazard to the environment. In contrast to the products of most transgenic plants currently available commercially (e.g., the insecticidal proteins from subspecies of Bt) that primarily target insects and other pests. These "pharms" are being genetically engineered to express products for use primarily in human beings. Consequently, these products constitute a class of compounds that is seldom found in natural habitats and that primarily target "higher level" eukaryotes. Hence, they are xenobiotics with respect to the environment, and their persistence in and effects on the environment have not been adequately studied and sober risk assessments on a case-by- case basis must be made before major releases of such transgenic organisms.

Structural Basis of Pore Formation by Mosquito-larvicidal Proteins from Bacillus thuringiensis

Abstract: The insecticidal character of the three-domain Cry  -endotoxins produced by Bacillus thuringiensis during sporulation is believed to be caused by their capability to generate lytic pores in the target larval midgut cell membranes. This review describes toxic mechanisms with emphasis on the structural basis of pore formation by two closely related dipteran-specific toxins, Cry4Aa and Cry4Ba, which are highly toxic to mosquito larvae. One proposed toxic mechanism via an "umbrella-like" structure involves membrane penetration and pore formation by the � 4-� 5 transmembrane hairpin. The lipid-induced  -conformation of  7 could possibly serve as a lipid anchor required for an efficient insertion of the pore-forming hairpin into the bilayer membrane. Though current electron crystallographic data are still inadequate to provide such critical insights into the structural details of the Cry toxin-induced pore architecture, this pivotal evidence clearly reveals that the 65-kDa active toxin in association with the lipid membrane could exist in at least two different trimeric conformations, implying the closed and open states of a functional pore.

The Intracellular Journey of Shiga Toxins

Abstract: The Shiga toxin family consists of Shiga toxin (Stx) that is produced as a virulence factor by Shigella dysenteriae, and the Shiga-like toxins produced by certain strains of enterohemorrhagic E. coli as well as by some other types of bacteria. Infection with bacteria producing these toxins is a threat to human health even in industrialized countries, as the initial diarrhea caused by the infection might be followed by a complication named hemolytic uremic syndrome. The Shiga toxins consist of a binding moiety that in most cases binds to the glycosphingolipid Gb3 on the surface of susceptible cells, and an A-moiety responsible for the toxic effect in the cytosol. In order to reach its cytosolic target, the toxin must be internalized and then transported via the retrograde pathway to the Golgi complex and further to the endoplasmic reticulum. From the endoplasmic reticulum the enzymatically active part of the A-moiety is translocated to the cytosol, and cellular protein synthesis is inhibited. Although the Shiga toxins are involved in disease, they may also be exploited for medical diagnosis and treatment. Interestingly, the toxin receptor, Gb3, has a limited expression in normal tissues, but is overexpressed in several types of cancer. Thus, the use of Shiga toxin, or the binding part of the toxin, has great potential in cancer diagnostics and treatment. Furthermore, studies of the various uptake mechanisms and intracellular transport pathways exploited by the toxins, provide important insight in basic cell biology processes.

Clostridium Botulinum C3 Exoenzyme: Rho-Inactivating Tool in Cell Biology and a Neurotrophic Agent

Abstract: C3 exoenzyme from Clostridium botulinum is the prototype of bacterial ADP-ribosyltransferases, which selectively modifies the Rho isoforms RhoA, RhoB and RhoC by covalent attachment of an ADP-ribose moiety. ADP- ribosylation results in inactivation of cellular functions of Rho. Because of its highly restricted substrate specificity, C3 is an established tool in cell biology; to this end C3 is applied as a cell-permeable chimeric toxin. C3 is superior to other molecular biology techniques such as siRNA or knock down approaches as RhoA inactivation or knock down is intrinsically associated with RhoB activation except after C3 treatment. RhoA plays an essential role in axonal growth and repair after neuronal injury. For therapeutic purposes cell- permeable C3 is now locally administered to treat spinal cord injury. Recently, it was reported that ADP-ribosyltransferase activity is not essential for the neurotrophic effect of C3 and that a peptidic fragment of C3 acts neurotrophic.

Synergy Between Aedes aegypti Trypsin Modulating Oostatic Factor and δ-Endotoxins

Abstract: Starved first instar Aedes aegypti larvae were 35-fold more sensitive to Bacillus thuringiensis subsp. israelensis (Bti) toxins than fed larvae. Feeding larvae Pichia pastoris yeast cells expressing tmfA (synthetic gene coding for the Trypsin Modulating Oostatic Factor of Ae. aegypti) together with Escherichia coli cells expressing Bti toxin genes (cry4Aa, cry11Aa, cyt1Aa and p20) indicate that TMOF and Cry toxins are synergisitic. tmfA was cloned and expressed in the cyanobacterium Anabaena PCC 7120 and the hormone was purified by HPLC and identified by ELISA. The amount of TMOF synthesized by Anabaena was low (0.5 - 1 μg in 10 8 cells). P. pastoris, which synthesizes high amounts of heterologous proteins in the presence of methanol and is readily consumed by mosquito larvae, was genetically engineered to produce more TMOF. Codon-optimized synthetic genes, cry11Aa- tmfA and gst-cry11Aa- tmfA, that were cloned into P. pastoris and fed to Ae. aegypti larvae caused 87.5% mortality in 5 days. GST (glutathione-S-transferase) enhanced the activity of Cry11A-TMOF and protected it from heat denaturation. Cell free extracts of recombinant P. pastoris cells killed 40% of tested 4 th instar larvae within 24 h, and mass spectra analysis confirmed that the recombinants synthesize Cry11Aa. This report shows for the first time that Cry toxins and TMOF are synergists to Ae. aegypti larvae when jointly fed or expressed in recombinant P. pastoris.

Insecticidal Properties and Chemical Composition of Conyza Aegyptiaca (L.) Oil: Studies on Two Dipterous Insect Pests

Abstract: The herb, Conyza aegyptiaca L., was subjected to hydrodistillation process to extract the essential oil from the whole dry plant material. Larvae and adult stages of the mosquito, Anopheles pharoensis, and the housefly, Musca domes- tica, were used as test model organisms representing two dipterous insect pests of medical importance. Under standard bioassay test methods, the LC50 of the oil accounted to 37.8 ppm and 0.087 mg/cm 2 against larvae and adults of An. pharoensis, respectively. These toxicity parameters were found to be 71.8 ppm as LC50 and 0.125 µg/insect as LD50 against larvae and adults of M. domestica, respectively. Using GC/MS analysis, we identified 19 compounds constituting ca. 97% of phytochemicals present in the oil, such as monoterpenes, sesquiterpenes and esters. Limonene constituted about 50% of the plant oil (48.79%), followed by (E) - β-Ocimene (8.66%), Germacrene D (7.54%) and β-pinene (6.91%). The occurrence of the other constituents ranged between 0.27% and 5.29%. It was concluded that the potency of C. aegyptiaca oil refers mainly to the presence of limonene. The findings of this study may encourage more research aiming at investi- gation of eco-friendly biopesticides based on botanical resources.

Staphylococcal and Streptococcal Superantigens: Basic Biology of Conserved Protein Toxins

Abstract: Staphylococcus aureus and Streptococcus pyogenes are gram-positive bacteria that possess great pathogenic potential in humans, causing numerous maladies such as arthritis, cutaneous infections, endocarditis, enterocolitis, food poisoning, pharyngitis, pneumonia, rheumatic fever, surgical site infections, and toxic shock. These prevalent pathogens produce various virulence factors that include the staphylococcal enterotoxins (SEs), toxic shock syndrome toxin-1 (TSST-1), and streptococcal pyrogenic exotoxins (SPEs). Minute (picomolar) amounts of these structurally-similar "superantigens" (SAgs) elicit high levels of proinflammatory cytokines and chemokines that can induce fever, hypotension, and lethal shock. In vitro and in vivo models have provided important tools for studying the biological effects of, and potential vaccines plus therapeutics against, these related protein toxins. This review will delve into the known physical and biological properties of the SEs, TSST-1, and SPEs. The reader will hopefully derive a general appreciation of these wonderfully-complex, structurally-similar toxins produced by S. aureus and S. pyogenes.

Glucosylation of Rho/Ras Proteins by Lethal Toxin – Implications of Actin Re-Organization and Apoptosis in C. Sordellii-Associated Disease

Abstract: Clostridium sordellii causes disease in livestock and life-threatening illnesses in humans. Pathogenic C. sordellii strains produce up to seven virulence factors, including lethal toxin (TcsL), hemorrhagic toxin, a hemolysin, a DNAse, a collagenase, and a lysolecithinase cell. TcsL exhibits an A-B toxin-like structure and enters its target cells by receptor-mediated endocytosis. Inside the, TcsL mono-glucosylates low molecular weight GTP-binding proteins of the Ras and Rho families. This article reviews recent progress for (i) re-enforcing (H/K/N)Ras glucosylation and subsequent inhibition of the phosphoinositide 3-kinase (PI3K) / Akt survival signalling pathway as the cause of TcsL-induced apoptotic cell death, and (ii) showing the critical nature of Rac1 glucosylation in the loss of epithelial and endothelial barrier function. Finally, the detection of TcsL-induced glucosylation of Rac1 and (H/K/N)Ras using glucosylation- sensitive antibodies is presented as a new method to track TcsL activity.

Role of Host Cell Chaperones in Cellular Uptake of Clostridium Botulinum C2 Toxin

Abstract: The binary C2 toxin from Clostridium botulinum consists of two separate proteins: the transport component C2IIa delivers the enzyme component C2I into the cytosol of eukaryotic host cells. In the cytosol, C2I mono-ADP- ribosylates actin, thereby inducing depolymerization of actin filaments resulting in delayed caspase-dependent cell death. The sophisticated cellular uptake mechanism of C2 toxin, in particular our new results regarding the role of host cell chaperones and protein-folding helper enzymes during intracellular membrane translocation of C2I, are focused upon in this minireview. We discovered earlier that translocation of C2I across endosomal membranes in mammalian cells depends on the chaperone activity of the heat shock protein Hsp90. Recently we have demonstrated that cyclosporin A (CsA), an inhibitor of peptidyl-prolyl cis/trans isomerase (PPIase) activity of cyclophilins, inhibited intoxication of various mammalian cell lines with C2 toxin. The underlying reason for this effect was the prevented uptake of C2I into the host cell cytosol. CsA, as well as a specific antibody against cyclophilin A, blocked the pH-dependent translocation of C2I-ADP- ribosyltransferase activity across membranes of intact cells and of partially-purified early endosomes in vitro. In conclusion, our results imply that the activities of Hsp90 and cyclophilin A are crucial for translocation of the C2I ADP- ribosyltransferase from early endosomes into the cytosol of mammalian cells. This is the first observation that a host cell PPIase, in concert with a heat shock protein, facilitates intracellular membrane translocation of a bacterial protein toxin.

Iota Toxin, S Toxin and CDT: Members of the Same Class of Clostridial Binary Toxins in Feces of Humans and Other Animals

Abstract: Some strains of Clostridium perfringens, Clostridium spiroforme and Clostridium difficile produce binary toxins known respectively as iota toxin, S toxin and CDT. Each toxin consists of two unlinked polypeptides (e.g. CDTa and CDTb) that only together have biological activity. Taking an historical perspective, we review the development and early use of assays employing the specific neutralization of a biological activity for the detection and quantification of binary toxin. The survey moves on to more recent immunological assays and culminates with a discussion of the relevance of binary toxin, especially CDT, in feces.