Pathogenesis of cell toxicity by plant toxins

Ribosome inactivating proteins (RIPs) are a family of plant proteins that depurinate the major rRNA, inhibiting the protein synthesis. RIPs are divided into type 1, single chain proteins with enzymatic activity, and type 2 RIPs (toxic and non-toxic), with the enzymatic chain linked to a binding chain. RIPs have been used alone or as toxic component of immunotoxins for experimental therapy of many diseases. The knowledge of cell death pathway(s) induced by RIPs could be useful for clarifying the mechanisms induced by RIPs and for designing specific immunotherapy. The topic of the current study was (i) the determination of the amino acid sequence of the type 2 RIP stenodactylin. The comparison with other RIPs showed that the A chain is related to other toxic type 2 RIPs. whereas the B chain is more related to the non-toxic type 2 RIPs. This latter result is surprising because stenodactylin is actually the most toxic type 2 RIP known; (ii) the study of the cell death mechanisms induced by stenodactylin in human neuroblastoma cells (NB100). High doses of stenodactylin can activate the effector caspases (perhaps through the DNA damage and/or intrinsic/extrinsic pathways) and also cause ROS generation. Low doses cause a caspase-dependent apoptosis, mainly via extrinsic pathway. Moreover, the activation of caspases precedes the inhibition of protein synthesis; (iii) the investigation of the cell death pathway induced by the non-toxic type 2 RIPs ebulin l and nigrin b. These RIPs demonstrated high enzymatic activity in a cell-free system, but they lack high cytotoxicity. These preliminary studies demonstrate that the cell death mechanism induced by the two non-toxic RIPs is partially caspase-dependent apoptosis, but other mechanisms seem to be involved

Clostridium difficile toxins facilitate bacterial colonization by modulating the fence and gate function of colonic epithelium

The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa.

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

Background Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences. Results About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein. An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins. Conclusions The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of C. inanitus appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.

An increase in serum tryptase even below 11.4 ng/mL may indicate a mast cell-mediated hypersensitivity reaction: a prospective study in Hymenoptera venom allergic patients

During a systemic hypersensitivity reaction (SR), an increase in serum tryptase compared to the baseline value is an indicator of mast cell activation, most often due to an IgE-mediated mechanism.

Venom composition and strategies in spiders: is everything possible?

This review on all spider venom components known by the end of 2010 bases on 1618 records for venom compounds from 174 spider species (= 0.41% of all known species) belonging to 32 families (= 29% of all existing spider families). Spiders investigated for venom research are either big (many mygalomorph species, Nephilidae, Ctenidae and Sparassidae) or medically important for humans (e.g. Loxosceles or Latrodectus species). Venom research widely ignored so far the two most species-rich families (Salticidae and Linyphiidae) and strongly neglected several other very abundant families (Araneidae, Lycosidae, Theridiidae, Thomisidae and Gnaphosidae). We grouped the known 1618 records for venom compounds into six categories: low molecular mass compounds (16 % of all compounds), acylpolyamines (11 %), linear peptides (6 %), cysteine-knotted mini-proteins (60 %), neurotoxic proteins (1 %) and enzymes (6 %). Low molecular mass compounds are known from many spider families and contain organic acids, nucleosides, nucleotides, amino acids, amines, polyamines, and some further substances, many of them acting as neurotransmitters. Acylpolyamines contain amino acids (Araneidae and Nephilidae) or not (several other families) and show a very high diversity within one species. Linear peptides, also called cytolytic, membranolytic or antimicrobial, exert a highly specific structure and are so far only known from Ctenidae, Lycosidae, Oxyopidae and Zodariidae. Cysteine-knotted mini-proteins represent the majority of venom compounds because research so far focused on them. They probably occur in most but not all spider families. Neurotoxic proteins so far are only known from theridiid spiders. Enzymes had been neglected for some time but meanwhile it becomes obvious that they play an important role in spider venoms. Sixteen enzymes either cleave polymers in the extracellular matrix or target phospholipids and related compounds in membranes. The overall structure of these compounds is given and the function, as far as it is known, is described. Since several of these component groups are presented in one average spider venom, we discuss the known interactions and synergisms and give reasons for such a functional redundancy. We also discuss main evolutionary pathways for spider venom compounds such as high variability among components of one group, synergistic interactions between cysteine-knotted mini-proteins and other components (low molecular mass compounds and linear peptides), change of function from ion-channel acting mini-proteins to cytolytic effects and replacement of mini-proteins by linear peptides, acylpolyamines, large proteins or enzymes. We also add first phylogenetic considerations.

The role of RTX toxins in host specificity of animal pathogenic Pasteurellaceae

RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary pathogens of the family of Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal in vitro in experimental studies. More recent studies have shown that the specific targets of most RTX toxins are leukocytes, with RTX toxins binding to the corresponding beta-subunit (CD18) of beta2 integrins and then exerting cytotoxic activity. After uptake by the target cell, at sub-lytic concentrations, some RTX toxins are transported to mitochondria and induce apoptosis. For several RTX toxins the binding to CD18 has been shown to be host specific and this seems to be the basis for the host range specificity of these RTX toxins. Observations on two very closely related species of the Pasteurellaceae family, Actinobacillus suis, a porcine pathogen particularly affecting suckling pigs, and Actinobacillus equuli subsp. haemolytica, which causes pyosepticaemia in new-born foals (sleepy foal disease), have revealed that they express different RTX toxins, named ApxI/II and Aqx, respectively. These RTX toxins are specifically cytotoxic for porcine and equine leukocytes, respectively. Furthermore, the ApxI and Aqx toxins of these species, when expressed in an isogenetic background in Escherichia coli, are specifically cytotoxic for leukocytes of their respective hosts. These data indicate the determinative role of RTX toxins in host specificity of pathogenic species of Pasteurellaceae.

A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

IgE to recombinant allergens Api m 1, Ves v 1, and Ves v 5 distinguish double sensitization from crossreaction in venom allergy

Diagnostic tests in patients with Hymenoptera venom allergy are frequently positive to venoms of both honey bee and wasp (Vespula). Component-resolved analysis with recombinant species-specific major allergens (rSSMA) may help to distinguish true double sensitization from crossreactivity.

Woman and Snake

Mixed Media 39 x 42"

Snake Goddess

Mixed Media 40 x 36"

Ophioluxin, a convulxin-like C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI

Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.

Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMkappa responsible for platelet agglutination in plasma and inducing signal transduction

Echicetin, a heterodimeric snake C-type lectin from Echis carinatus, is known to bind specifically to platelet glycoprotein (GP)Ib. We now show that, in addition, it agglutinates platelets in plasma and induces platelet signal transduction. The agglutination is caused by binding to a specific protein in plasma. The protein was isolated from plasma and shown to cause platelet agglutination when added to washed platelets in the presence of echicetin. It was identified as immunoglobulin Mkappa (IgMkappa) by peptide sequencing and dot blotting with specific heavy and light chain anti-immunoglobulin reagents. Platelet agglutination by clustering echicetin with IgMkappa induced P-selectin expression and activation of GPIIb/IIIa as well as tyrosine phosphorylation of several signal transduction molecules, including p53/56(LYN), p64, p72(SYK), p70 to p90, and p120. However, neither ethylenediaminetetraacetic acid nor specific inhibition of GPIIb/IIIa affected platelet agglutination or activation by echicetin. Platelet agglutination and induction of signal transduction could also be produced by cross-linking biotinylated echicetin with avidin. These data indicate that clustering of GPIb alone is sufficient to activate platelets. In vivo, echicetin probably activates platelets rather than inhibits platelet activation, as previously proposed, accounting for the observed induction of thrombocytopenia.