Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Quantification of snake venom proteomes by mass spectrometry-considerations and perspectives.

The advent of soft ionization mass spectrometry-based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global-integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of "an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available." This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label-free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term "non-model" organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review-essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof-of-concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.

Venom-microbiomics of eight species of Neotropical spiders from the Theraphosidae family.

AIM: Tarantulas are one of the largest predatory arthropods in tropical regions. Tarantulas though not lethal to humans, their venomous bite kills small animals and insect upon which they prey. To understand the abiotic and biotic components involved in Neotropical tarantula bites, we conducted a venom-microbiomics study in eight species from Costa Rica. METHODS AND RESULTS: We determined that the toxin profiles of tarantula venom are highly diverse using shotgun proteomics; the most frequently encountered toxins were ω-Ap2 toxin, neprilysin-1, and several teraphotoxins. Through culture-independent and culture-dependent methods, we determined the microbiota present in the venom and excreta to evaluate the presence of pathogens that could contribute to primary infections in animals, including humans. The presence of opportunistic pathogens with hemolytic activity was observed, with a prominence of Stenotrophomonas in the venoms. Other bacteria found in venoms and excreta with hemolytic activity included members of the genera Serratia, Bacillus, Acinetobacter, Microbacterium, and Morganella. CONCLUSIONS: Our data shed light on the venom- and gut-microbiome associated with Neotropical tarantulas. This information may be useful for treating bites from these arthropods in both humans and farm animals, while also providing insight into the toxins and biodiversity of this little-explored microenvironment.

Skeletal muscle fiber hypercontraction induced by Bothrops asper myotoxic phospholipases A2 ex vivo does not involve a direct action on the contractile apparatus.

Myonecrosis is a frequent clinical manifestation of envenomings by Viperidae snakes, mainly caused by the toxic actions of secreted phospholipase A2 (sPLA2) enzymes and sPLA2-like homologs on skeletal muscle fibers. A hallmark of the necrotic process induced by these myotoxins is the rapid appearance of hypercontracted muscle fibers, attributed to the massive influx of Ca2+ resulting from cell membrane damage. However, the possibility of myotoxins having, in addition, a direct effect on the contractile machinery of skeletal muscle fibers when internalized has not been investigated. This question is here addressed by using an ex vivo model of single-skinned muscle fibers, which lack membranes but retain an intact contractile apparatus. Rabbit psoas skinned fibers were exposed to two types of myotoxins of Bothrops asper venom: Mt-I, a catalytically active Asp49 sPLA2 enzyme, and Mt-II, a Lys49 sPLA2-like protein devoid of phospholipolytic activity. Neither of these myotoxins affected the main parameters of force development in striated muscle sarcomeres of the skinned fibers. Moreover, no microscopical alterations were evidenced after their exposure to Mt-I or Mt-II. In contrast to the lack of effects on skinned muscle fibers, both myotoxins induced a strong hypercontraction in myotubes differentiated from murine C2C12 myoblasts, with drastic morphological alterations that reproduce those described in myonecrotic tissue in vivo. As neither Mt-I nor Mt-II showed direct effects upon the contractile apparatus of skinned fibers, it is concluded that the mechanism of hypercontraction triggered by both myotoxins in patients involves indirect effects, i.e., the large cytosolic Ca2+ increase after sarcolemma permeabilization.

Alterations of the skeletal muscle contractile apparatus in necrosis induced by myotoxic snake venom phospholipases A2: a mini-review.

Skeletal muscle necrosis is a common clinical manifestation of snakebite envenoming. The predominant myotoxic components in snake venoms are catalytically-active phospholipases A2 (PLA2) and PLA2 homologs devoid of enzymatic activity, which have been used as models to investigate various aspects of muscle degeneration. This review addresses the changes in the contractile apparatus of skeletal muscle induced by these toxins. Myotoxic components initially disrupt the integrity of sarcolemma, generating a calcium influx that causes various degenerative events, including hypercontraction of myofilaments. There is removal of specific sarcomeric proteins, owing to the hydrolytic action of muscle calpains and proteinases from invading inflammatory cells, causing an initial redistribution followed by widespread degradation of myofibrillar material. Experiments using skinned cardiomyocytes and skeletal muscle fibers show that these myotoxins do not directly affect the contractile apparatus, implying that hypercontraction is due to cytosolic calcium increase secondary to sarcolemmal damage. Such drastic hypercontraction may contribute to muscle damage by generating mechanical stress and further sarcolemmal damage.

SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance.

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A2 homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 µM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.

First Insights into the Venom Composition of Two Ecuadorian Coral Snakes.

Micrurus is a medically relevant genus of venomous snakes composed of 85 species. Bites caused by coral snakes are rare, but they are usually associated with very severe and life-threatening clinical manifestations. Ecuador is a highly biodiverse country with a complex natural environment, which is home to approximately 20% of identified Micrurus species. Additionally, it is on the list of Latin American countries with the highest number of snakebites. However, there is no local antivenom available against the Ecuadorian snake venoms, and the biochemistry of these venoms has been poorly explored. Only a limited number of samples collected in the country from the Viperidae family were recently characterised. Therefore, this study addressed the compositional patterns of two coral snake venoms from Ecuador, M. helleri and M. mipartitus, using venomics strategies, integrating sample fractionation, gel electrophoresis, and mass spectrometry. Chromatographic and electrophoretic profiles of these snake venoms revealed interspecific variability, which was ascertained by mass spectrometry. The two venoms followed the recently recognised dichotomic toxin expression trends displayed by Micrurus species: M. helleri venom contains a high proportion (72%) of phospholipase A2, whereas M. mipartitus venom is dominated by three-finger toxins (63%). A few additional protein families were also detected in these venoms. Overall, these results provide the first comprehensive views on the composition of two Ecuadorian coral snake venoms and expand the knowledge of Micrurus venom phenotypes. These findings open novel perspectives to further research the functional aspects of these biological cocktails of PLA2s and 3FTxs and stress the need for the preclinical evaluation of the currently used antivenoms for therapeutic purposes in Ecuador.

What's in a mass?

This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.

Danger in the Canopy. Comparative Proteomics and Bioactivities of the Venoms of the South American Palm Pit Viper Bothrops bilineatus Subspecies bilineatus and smaragdinus and Antivenomics of B. b. bilineatus (Rondônia) Venom against the Brazilian Pentabothropic Antivenom.

We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34-40% of the total venom proteins) and PI (8-18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8-10%); bradykinin-potentiating-like peptides (BBPs, 10.7-15%); snake venom serine proteinases (SVSP, 5.5-14%); C-type lectin-like proteins (CTL, 3-10%); phospholipases A2 (PLA2, 2.8-7.6%); cysteine-rich secretory proteins (CRISP, 0.9-2.8%); l-amino acid oxidases (LAO, 0.9-5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops ″jararaca″ and ″taeniatus″ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.

Immunological cross-recognition and neutralization studies of Micrurus mipartitus and Micrurus dumerilii venoms by two therapeutic equine antivenoms.

New world Coral snakes comprise 82 species of medical importance distributed from southeastern United States to Argentina. In Colombia, Micrurus mipartitus and M. dumerilii are responsible for most coral snakebite accidents. Although infrequent, the severity of these envenomings, as well as the limited information available on the neutralizing coverage of commercially available antivenoms, underscores the need to perform studies to assess the cross-neutralizing ability of these life-saving immunobiologicals. In the present work, we evaluated the cross-recognition and neutralization ability of two equine therapeutic antivenoms: PROBIOL and SAC-ICP. PROBIOL antivenom showed cross-recognition towards both M. mipartitus and M. dumerilii venoms, with a significantly higher binding to the latter in both whole-venom ELISA and fractionated-venom immunoprofiling. In contrast, SAC-ICP antivenom cross-recognized M. dumerilii venom, but not that of M. mipartitus. Lethality of M. dumerilii venom was neutralized by both antivenoms, with a slightly higher potency for the SAC-ICP antivenom. However, the lethality of M. mipartitus venom was not neutralized by any of the two antivenoms. Results uncover the need to include M. mipartitus venom, or its most relevant toxins, in the production of coral snake antivenoms to be used in Colombia, to assure the neutralizing coverage for this species.

Potent virucidal activity against Flaviviridae of a group IIA phospholipase A2 isolated from the venom of Bothrops asper.

Secreted phospholipase A2 (sPLA2) molecules are small, calcium-dependent enzymes involved in many biological processes. Viperid venoms possess gIIA sPLA2s and sPLA2-like proteins, both having homology to human gIIA sPLA2, an innate immunity enzyme. We evaluated the antiviral action of Mt-I (catalytically-active sPLA2) and Mt-II (catalytically-inactive variant) isolated from the venom of Bothrops asper, against a diverse group of viruses. Yellow Fever and Dengue (enveloped) viruses were highly susceptible to inactivation by the snake proteins, in contrast to Sabin (non-enveloped; Polio vaccine strain), and Influenza A, Herpes simplex 1 and 2, and Vesicular Stomatitis (enveloped) viruses. Titration of the antiviral effect against Dengue virus revealed Mt-I to be highly potent (IC50 0.5-2 ng/mL), whereas Mt-II was 1000-fold weaker. This large difference suggested a requirement for PLA2 activity, which was confirmed by chemical inactivation of Mt-I. A synthetic peptide representing the membrane-disrupting region of Mt-II, previously shown to have bactericidal effect, lacked antiviral action, suggesting that the weak virucidal effect observed for Mt-II is likely caused by contamination with traces of Mt-I. On the other hand, Mt-I was demonstrated to act by a direct virucidal mechanism prior to infection, and not by an independent effect on host cells, either pretreated, or exposed to Mt-I after virus infection. Interestingly, DENV2 propagated in mosquito cells was much more sensitive to the action of Mt-I, compared to human cell-propagated virus. Therefore, differences in envelope membrane composition may be crucially involved in the observed virucidal action of PLA2 enzymes.

Proteogenomic analysis of the Clostridium difficile exoproteome reveals a correlation between phylogenetic distribution and virulence potential.

C. difficile induces antibiotic-associated diarrhea due to the action of two secreted toxins, TcdA and TcdB. A considerable range of virulence among C. difficile strains has been widely reported. During a hospital outbreak, 46 isolates were collected that belonged to different genotypes. Of those, the majority corresponded to two virulent strains, the globally distributed Sequence Type 1 (ST1)_North American Pulsotype 1 (NAP1) and the endemic ST54_NAPCR1 genotypes, respectively. Whereas the virulence of the latter has been attributed to increased secretion of toxins and production of a highly cytotoxic TcdB, these characteristics do not explain the increased lethality of the former. We undertook a proteomic comparative approach of the isolates participating in the outbreak to look for proteins present in the exoproteome of the ST1_NAP1and ST54_NAPCR1 strains. We used a low virulent ST2_NAP4 strain isolated also in the outbreak as control. Dendrograms constructed using the exoproteomes of the strains were very similar to those created using genomic information, suggesting an association between secreted proteins and relative virulence of the strains. By 2D electrophoresis and mass spectrometry it was found that approximately half of the proteins are shared among strains of different genotypes. From the identified proteins, the surface-located SlpA draw our attention due to its detection in ST54_NAPCR1 exoproteomes. Biochemical analysis indicated that the processing of SlpA is different in the ST54_NAPCR1 strain and confirmed that this strain secretes more SlpA than its counterparts. Furthermore, SlpA from the ST54_NAPCR1 strain exerted an increased proinflammatory activity. Altogether, these results indicate that the exoproteome composition correlates with the C. difficile genotype and suggest that particular proteins secreted by some strains could synergize with the effects of TcdA and TcdB increasing their virulence.

A Secreted Phospholipase A2 Induces Formation of Smooth Muscle Foam Cells Which Transdifferentiate to Macrophage-Like State.

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-γ and -ß/δ. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.

Depletion of Complement Enhances the Clearance of Brucella abortus in Mice.

Brucellosis is a bacterial disease of animals and humans. Brucella abortus barely activates the innate immune system at the onset of infection, and this bacterium is resistant to the microbicidal action of complement. Since complement stands as the first line of defense during bacterial invasions, we explored the role of complement in B. abortus infections. Brucella abortus-infected mice depleted of complement with cobra venom factor (CVF) showed the same survival rate as mice in the control group. The complement-depleted mice readily eliminated B. abortus from the spleen and did so more efficiently than the infected controls after 7 days of infection. The levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-6 (IL-6) remained within background levels in complement-depleted B. abortus-infected mice. In contrast, the levels of the immune activator cytokine gamma interferon and the regulatory cytokine IL-10 were significantly increased. No significant histopathological changes in the liver and spleen were observed between the complement-depleted B. abortus-infected mice and the corresponding controls. The action exerted by Brucella on the immune system in the absence of complement may correspond to a broader phenomenon that involves several components of innate immunity.

A Snake Venom-Secreted Phospholipase A2 Induces Foam Cell Formation Depending on the Activation of Factors Involved in Lipid Homeostasis.

MT-III, a snake venom GIIA sPLA2, which shares structural and functional features with mammalian GIIA sPLA2s, activates macrophage defense functions including lipid droplet (LDs) formation, organelle involved in both lipid metabolism and inflammatory processes. Macrophages (MΦs) loaded with LDs, termed foam cells, characterize early blood vessel fatty-streak lesions during atherosclerosis. However, the factors involved in foam cell formation induced by a GIIA sPLA2 are still unknown. Here, we investigated the participation of lipid homeostasis-related factors in LD formation induced by MT-III in macrophages. We found that MT-III activated PPAR-γ and PPAR-ß/δ and increased the protein levels of both transcription factors and CD36 in macrophages. Pharmacological interventions evidenced that PPAR-γ, PPAR-ß/δ, and CD36 as well as the endoplasmic reticulum enzymes ACAT and DGAT are essential for LD formation. Moreover, PPAR-ß/δ, but not PPAR-γ, is involved in MT-III-induced PLIN2 protein expression, and both PPAR-ß/δ and PPAR-γ upregulated CD36 protein expression, which contributes to MT-III-induced COX-2 expression. Furthermore, production of 15-d-PGJ2, an activator of PPARs, induced by MT-III, was dependent on COX-1 being LDs an important platform for generation of this mediator.

A constant area monolayer method to assess optimal lipid packing for lipolysis tested with several secreted phospholipase A2.

We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Πopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN·m(-1) and 10 mN·m(-1), termed here as LR20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.

N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils.

Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution.

Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms.

We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.

A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms.

Envenomation via snakebites is an important public health problem in many tropical and subtropical countries that, in addition to mortality, can result in permanent sequelae as a consequence of local tissue damage, which represents a major challenge to antivenom therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a leading role in the complex pathogenesis of skeletal muscle necrosis, nevertheless their precise mechanism of action is only partially understood. Recently, detailed structural information has been obtained for more than twenty different members of the PLA2-like myotoxin subfamily. In this review, we integrate the available structural, biochemical and functional data on these toxins and present a comprehensive hypothesis for their myotoxic mechanism. This process involves an allosteric transition and the participation of two independent interaction sites for docking and disruption of the target membrane, respectively, leading to a five-step mechanism of action. Furthermore, recent functional and structural studies of these toxins complexed with ligands reveal diverse neutralization mechanisms that can be classified into at least three different groups. Therefore, the data summarized here for the PLA2-like myotoxins could provide a useful molecular basis for the search for novel neutralizing strategies to improve the treatment of envenomation by viperid snakes.

An Asp49 phospholipase A2 from snake venom induces cyclooxygenase-2 expression and prostaglandin E2 production via activation of NF-κB, p38MAPK, and PKC in macrophages.

Phospholipases A2 (PLA2) are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PG)E2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2). Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.