Past, Present, and Future of Naturally Occurring Antimicrobials Related to Snake Venoms.

This review focuses on proteins and peptides with antimicrobial activity because these biopolymers can be useful in the fight against infectious diseases and to overcome the critical problem of microbial resistance to antibiotics. In fact, snakes show the highest diversification among reptiles, surviving in various environments; their innate immunity is similar to mammals and the response of their plasma to bacteria and fungi has been explored mainly in ecological studies. Snake venoms are a rich source of components that have a variety of biological functions. Among them are proteins like lectins, metalloproteinases, serine proteinases, L-amino acid oxidases, phospholipases type A2, cysteine-rich secretory proteins, as well as many oligopeptides, such as waprins, cardiotoxins, cathelicidins, and ß-defensins. In vitro, these biomolecules were shown to be active against bacteria, fungi, parasites, and viruses that are pathogenic to humans. Not only cathelicidins, but all other proteins and oligopeptides from snake venom have been proteolyzed to provide short antimicrobial peptides, or for use as templates for developing a variety of short unnatural sequences based on their structures. In addition to organizing and discussing an expressive amount of information, this review also describes new ß-defensin sequences of Sistrurus miliarius that can lead to novel peptide-based antimicrobial agents, using a multidisciplinary approach that includes sequence phylogeny.

Past, present, and future of naturally occurring antimicrobials related to snake venoms

This review focuses on proteins and peptides with antimicrobial activity because these biopolymers can be useful in the fight against infectious diseases and to overcome the critical problem of microbial resistance to antibiotics. In fact, snakes show the highest diversification among reptiles, surviving in various environments; their innate immunity is similar to mammals and the response of their plasma to bacteria and fungi has been explored mainly in ecological studies. Snake venoms are a rich source of components that have a variety of biological functions. Among them are proteins like lectins, metalloproteinases, serine proteinases, L-amino acid oxidases, phospholipases type A2, cysteine-rich secretory proteins, as well as many oligopeptides, such as waprins, cardiotoxins, cathelicidins, and β-defensins. In vitro, these biomolecules were shown to be active against bacteria, fungi, parasites, and viruses that are pathogenic to humans. Not only cathelicidins, but all other proteins and oligopeptides from snake venom have been proteolyzed to provide short antimicrobial peptides, or for use as templates for developing a variety of short unnatural sequences based on their structures. In addition to organizing and discussing an expressive amount of information, this review also describes new β-defensin sequences of Sistrurus miliarius that can lead to novel peptide-based antimicrobial agents, using a multidisciplinary approach that includes sequence phylogeny.

Antimicrobial activity of snake β-defensins and derived peptides

β-defensins are antimicrobial peptides presenting in vertebrate animals. They participate in innate immunity, but little is known about them in reptiles, including snakes. Although several β-defensin genes were described in Brazilian snakes, their function is still unknown. The peptide sequence from these genes was deduced, and synthetic peptides (with approximately 40 amino acids and derived peptides) were tested against pathogenic bacteria and fungi using microbroth dilution assays. The linear peptides, derived from β-defensins, were designed applying the bioisosterism strategy. The linear β-defensins were more active against Escherichia coli, Micrococcus luteus, Citrobacter freundii, and Staphylococcus aureus. The derived peptides (7–14 mer) showed antibacterial activity against those bacteria and on Klebsiella pneumoniae. Nonetheless, they did not present activity against Candida albicans, Cryptococcus neoformans, Trychophyton rubrum, and Aspergillus fumigatus showing that the cysteine substitution to serine is deleterious to antifungal properties. Tryptophan residue showed to be necessary to improve antibacterial activity. Even though the studied snake β-defensins do not have high antimicrobial activity, they proved to be attractive as template molecules for the development of antibiotics.

The absence of thrombin-like activity in Bothrops erythromelas venom is due to the deletion of the snake venom thrombin-like enzyme gene.

Snake venom thrombin-like enzymes (SVTLEs) are serine proteinases that clot fibrinogen. SVTLEs are distributed mainly in venoms from snakes of the Viperidae family, comprising venomous pit viper snakes. Bothrops snakes are distributed throughout Central and South American and are responsible for most venomous snakebites. Most Bothrops snakes display thrombin-like activity in their venoms, but it has been shown that some species do not present it. In this work, to understand SVTLE polymorphism in Bothrops snake venoms, we studied individual samples from two species of medical importance in Brazil: Bothrops jararaca, distributed in Southeastern Brazil, which displays coagulant activity on plasma and fibrinogen, and Bothrops erythromelas, found in Northeastern Brazil, which lacks direct fibrinogen coagulant activity but shows plasma coagulant activity. We tested the coagulant activity of venoms and the presence of SVTLE genes by a PCR approach. The SVTLE gene structure in B. jararaca is similar to the Bothrops atrox snake, comprising five exons. We could not amplify SVTLE sequences from B. erythromelas DNA, except for a partial pseudogene. These genes underwent a positive selection in some sites, leading to an amino acid sequence diversification, mostly in exon 2. The phylogenetic tree constructed using SVTLE coding sequences confirms that they are related to the chymotrypsin/kallikrein family. Interestingly, we found a B. jararaca specimen whose venom lacked thrombin-like activity, and its gene sequence was a pseudogene with SVTLE structure, presenting nonsense and frameshift mutations. Our results indicate an association of the lack of thrombin-like activity in B. jararaca and B. erythromelas venoms with mutations and deletions of snake venom thrombin-like enzyme genes.

Antimicrobial Activity of Snake ß-Defensins and Derived Peptides.

ß-defensins are antimicrobial peptides presenting in vertebrate animals. They participate in innate immunity, but little is known about them in reptiles, including snakes. Although several ß-defensin genes were described in Brazilian snakes, their function is still unknown. The peptide sequence from these genes was deduced, and synthetic peptides (with approximately 40 amino acids and derived peptides) were tested against pathogenic bacteria and fungi using microbroth dilution assays. The linear peptides, derived from ß-defensins, were designed applying the bioisosterism strategy. The linear ß-defensins were more active against Escherichia coli, Micrococcus luteus, Citrobacter freundii, and Staphylococcus aureus. The derived peptides (7-14 mer) showed antibacterial activity against those bacteria and on Klebsiella pneumoniae. Nonetheless, they did not present activity against Candida albicans, Cryptococcus neoformans, Trychophyton rubrum, and Aspergillus fumigatus showing that the cysteine substitution to serine is deleterious to antifungal properties. Tryptophan residue showed to be necessary to improve antibacterial activity. Even though the studied snake ß-defensins do not have high antimicrobial activity, they proved to be attractive as template molecules for the development of antibiotics.

The absence of thrombin-like activity in Bothrops erythromelas venom is due to the deletion of the snake venom thrombin-like enzyme gene

Snake venom thrombin-like enzymes (SVTLEs) are serine proteinases that clot fibrinogen. SVTLEs are distributed mainly in venoms from snakes of the Viperidae family, comprising venomous pit viper snakes. Bothrops snakes are distributed throughout Central and South American and are responsible for most venomous snakebites. Most Bothrops snakes display thrombin-like activity in their venoms, but it has been shown that some species do not present it. In this work, to understand SVTLE polymorphism in Bothrops snake venoms, we studied individual samples from two species of medical importance in Brazil: Bothrops jararaca, distributed in Southeastern Brazil, which displays coagulant activity on plasma and fibrinogen, and Bothrops erythromelas, found in Northeastern Brazil, which lacks direct fibrinogen coagulant activity but shows plasma coagulant activity. We tested the coagulant activity of venoms and the presence of SVTLE genes by a PCR approach. The SVTLE gene structure in B. jararaca is similar to the Bothrops atrox snake, comprising five exons. We could not amplify SVTLE sequences from B. erythromelas DNA, except for a partial pseudogene. These genes underwent a positive selection in some sites, leading to an amino acid sequence diversification, mostly in exon 2. The phylogenetic tree constructed using SVTLE coding sequences confirms that they are related to the chymotrypsin/kallikrein family. Interestingly, we found a B. jararaca specimen whose venom lacked thrombin-like activity, and its gene sequence was a pseudogene with SVTLE structure, presenting nonsense and frameshift mutations. Our results indicate an association of the lack of thrombin-like activity in B. jararaca and B. erythromelas venoms with mutations and deletions of snake venom thrombin-like enzyme genes.

Factor XII-Deficient Chicken Plasma as a Useful Target for Screening of Pro- and Anticoagulant Animal Venom Toxins.

The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [≥ 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to ≥ 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution.

Factor XII-deficient chicken plasma as a useful target for screening of pro- and anticoagulant animal venom toxins

The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [= 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to = 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution

Factor XII-deficient chicken plasma as a useful target for screening of pro- and anticoagulant animal venom toxins

The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [= 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to = 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution

Beta-defensin genes of the Colubridae snakes Phalotris mertensi, Thamnodynastes hypoconia, and T. strigatus.

ß-Defensins are cationic antimicrobial peptides showing little sequence similarity but highly conserved tertiary structure stabilized by a six-cysteines-motif. Using a PCR approach, we described ß-defensin sequences with two exons in three species of Colubridae snakes with high sequence similarity between them. The deduced amino acid sequence presented the characteristics of ß-defensin family. The phylogenetic analysis using ß-defensin coding sequences of different snakes grouped them in two main branches: genes organized in three or two exons.

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED50 and LD50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a P-neurotoxin phospholipase A(2)-like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED50) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro-or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control.

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED50 and LD50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a P-neurotoxin phospholipase A(2)-like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED50) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro-or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control.

Beta-defensin genes of the Colubridae snakes Phalotris mertensi, Thamnodynastes hypoconia, and T. strigatus

beta-Defensins are cationic antimicrobial peptides showing little sequence similarity but highly conserved tertiary structure stabilized by a six-cysteines-motif. Using a PCR approach, we described beta-defensin sequences with two exons in three species of Colubridae snakes with high sequence similarity between them. The deduced amino acid sequence presented the characteristics of beta-defensin family. The phylogenetic analysis using beta-defensin coding sequences of different snakes grouped them in two main branches: genes organized in three or two exons.

HSP70 expression in Biomphalaria glabrata snails exposed to cadmium.

In this study, the effects of the heavy metal cadmium on the stress protein HSP70 are investigated in freshwater mollusks Biomphalaria glabrata. Adult snails were exposed for 96h to CdCl2 at concentrations ranging from 0.09 to 0.7mgL-1 (LC50/96h=0.34 (0.30-0.37). Time and concentration-dependent increases in the expression of HSP70 were observed at sub-lethal levels in the immunoblotting assay. Further, an increased survival to a lethal heat shock was observed in animals pre-exposed to a nonlethal concentration of cadmium, evidencing the induction of acquired tolerance. The present study demonstrated the inducibility of B. glabrata HSP70 by cadmium, a relevant environmental contaminant, at non-lethal levels, providing evidences that the assessment of HSP70 in B. glabrata can be regarded as a suitable biomarker for ecotoxicological studies.

HSP70 expression in Biomphalaria glabrata snails exposed to cadmium

In this study, the effects of the heavy metal cadmium on the stress protein HSP70 are investigated in freshwater mollusks Biomphalaria glabrata. Adult snails were exposed for 96 h to CdCl2 at concentrations ranging from 0.09 to 0.7 mg L-1 (LC50/96 (h) = 0.34 (0.30-0.37). Time and concentration-dependent increases in the expression of HSP70 were observed at sub-lethal levels in the immunoblotting assay. Further, an increased survival to a lethal heat shock was observed in animals pre-exposed to a nonlethal concentration of cadmium, evidencing the induction of acquired tolerance. The present study demonstrated the inducibility of B. glabrata HSP70 by cadmium, a relevant environmental contaminant, at non-lethal levels, providing evidences that the assessment of HSP70 in B. glabrata can be regarded as a suitable biomarker for ecotoxicological studies.

Proteomic analysis of the rare Uracoan rattlesnake Crotalus vegrandis venom: Evidence of a broad arsenal of toxins.

The investigation of venoms has many clinical, pharmacological, ecological and evolutionary outcomes. The Crotalus spp. venom can cause hemorrhage, neurotoxicity, myotoxicity, coagulopathy and hypotension. Although neurotoxicity and hemorrhage usually does not occur for the same species, the rare Venezuelan species Crotalus vegrandis presents both characteristic. Different from the other species it has a restricted ecological niche and geographical distribution. Nevertheless, it has a raising medical importance as this rattlesnake population is increasing. Few works describe its neurotoxic and hemorrhagic features, but other toxins might play an important role in envenomation. We combined proteomic methods to identify for the first time the main components of it venom: 2D SDS-PAGE and gel-filtration chromatography for protein mixture decomplexation; LC-MS(2) of low molecular mass fractions and tryptic peptides; bioinformatic identification of toxin families and specific protein species based on unique peptide analysis and sequence database enriched with species-specific venom gland transcripts; and finally polyclonal anti-crotamine Western-blotting. Our results point to a broad arsenal of toxins in C. vegrandis venom: PIII and PII metalloproteases, crotoxin subunits, other phospholipases, isoforms of serine proteases and lectins, l-amino-acid oxidase, nerve growth factor, as well as other less abundant toxins.

Interaction of the rattlesnake toxin crotamine with model membranes.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. A common gene ancestry and structural similarity with the antimicrobial ß-defensins (identical disulfide bond pattern and highly positive net charge) suggested potential antimicrobial activities for this snake toxin. Although crotamine demonstrated low activity against both Gram-positive and Gram-negative bacteria, a pronounced antifungal activity was observed against Candida spp., Trichosporon spp., and Cryptococcus neoformans. Crotamine's selective antimicrobial properties, with no observable hemolytic activity, stimulated us to evaluate the potential applications of this polypeptide as an antiyeast or candicidal agent for medical and industrial application. Aiming to understand the mechanism(s) of action underlying crotamine antimicrobial activity and its selectivity for fungi, we present herein studies using membrane model systems (i.e., large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs), with different phospholipid compositions. We show here that crotamine presents a higher lytic activity on negatively charged membranes compared with neutral membranes, with or without cholesterol or ergosterol content. The vesicle burst was not preceded by membrane permeabilization as is generally observed for pore forming peptides. Although such a property of disrupting lipid membranes is very important to combat multiresistant fungi, no inhibitory activity was observed for crotamine against biofilms formed by several Candida spp. strains, except for a limited effect against C. krusei biofilm.

Phylogenetic analysis of ß-defensin-like genes of Bothrops, Crotalus and Lachesis snakes.

Defensins are components of the vertebrate innate immune system; they comprise a diverse group of small cationic antimicrobial peptides. Among them, ß-defensins have a characteristic ß-sheet-rich fold plus six conserved cysteines with particular spacing and intramolecular bonds. They have been fully studied in mammals, but there is little information about them in snakes. Using a PCR approach, we described 13 ß-defensin-like sequences in Bothrops and Lachesis snakes. The genes are organized in three exons and two introns, with exception of B.atrox_defensinB_01 which has only two exons. They show high similarities in exon 1, intron 1 and intron 2, but exons 2 and 3 have undergone accelerated evolution. The theoretical translated sequences encode a pre-ß-defensin-like molecule with a conserved signal peptide and a mature peptide. The signal peptides are leucine-rich and the mature ß-defensin-like molecules have a size around 4.5 kDa, a net charge from +2 to +11, and the conserved cysteine motif. Phylogenetic analysis was done using maximum parsimony, maximum likelihood and Bayesian analyses, and all resulted in similar topologies with slight differences. The genus Bothrops displayed two separate lineages. The reconciliation of gene trees and species tree indicated eight to nine duplications and 23 to 29 extinctions depending on the gene tree used. Our results together with previously published data indicate that the ancestral ß-defensin-like gene may have three exons in vertebrates and that their evolution occurred according to a birth-and-death model.