Main active compounds present in scorpion and spider venoms and

14 Pages·2015·1.22 MB·English

by  Francielle A. Cordeiro

Cordeiroetal.JournalofVenomousAnimalsandToxins
includingTropicalDiseases (2015) 21:24
DOI10.1186/s40409-015-0028-5
REVIEW Open Access
Arachnids of medical importance in Brazil:
main active compounds present in
scorpion and spider venoms and tick saliva
Francielle A. Cordeiro, Fernanda G. Amorim, Fernando A. P. Anjolette and Eliane C. Arantes*
Abstract
Arachnida is the largest class among the arthropods, constituting over 60,000 described species (spiders, mites,
ticks, scorpions, palpigrades, pseudoscorpions, solpugids and harvestmen). Many accidents are caused by
arachnids, especially spiders and scorpions, while some diseases can be transmitted by mites and ticks. These
animals are widely dispersed in urban centers due to the large availability of shelter and food, increasing the
incidence of accidents. Several protein and non-protein compounds present in the venom and saliva of these
animals are responsible for symptoms observed in envenoming, exhibiting neurotoxic, dermonecrotic and
hemorrhagic activities. The phylogenomic analysis from the complementary DNA of single-copy nuclear
protein-coding genes shows that these animals share some common protein families known as neurotoxins,
defensins, hyaluronidase, antimicrobial peptides, phospholipases and proteinases. This indicates that the
venoms from these animals may present components with functional and structural similarities. Therefore, we
described in this review the main components present in spider and scorpion venom as well as in tick saliva,
since they have similar components. These three arachnids are responsible for many accidents of medical
relevance in Brazil. Additionally, this study shows potential biotechnological applications of some components
with important biological activities, which may motivate the conducting of further research studies on their
action mechanisms.
Keywords: Arachnid toxins, Scorpion venom, Spider venom, Tick saliva
Background reviewwillfocusonthemainBrazilianvenomousanimals
Envenomings are considered a neglected disease by the oftheArachnidaclassbelongingtoScorpionida, Araneae,
World Health Organization [1] and constitute a public Ixodidae orders as well as on the aspects related to enve-
health problem, especially in tropical countries. The ani- noming caused by these animals and their venom/saliva
mals responsible for such accidents possess an apparatus composition, highlighting the components of scientific
associated with a venom gland that is able to produce and medical interest.
a mixture rich in toxic and nontoxic components [2]. Thephylogenomicanalysisofthenuclearprotein-coding
Among the most studied arthropod venoms are those sequences from arthropod species suggests a common
from scorpions, spiders and ticks, belonging to the origin in the venom systems of scorpions, spiders and
phylum Arthropoda, class Arachnida, which correspond to ticks [5, 6]. Specifically, catabolite activator protein (CAP),
the purpose of this review. They are widely dispersed in defensins, hyaluronidase, Kunitz-like peptides (serine pro-
urban centers due to the large availability of shelter and teinase inhibitor), neurotoxins, lectins and phospholipase
food, which facilitates their reproduction and consequently areexamplesofcompoundssharedbytheseanimals(Fig.1).
increases the number of accidents [3, 4]. Therefore, this Some compounds such as alanine-valine-isoleucine-threo-
nine protein (AVIT protein) and sphingomyelinase have
been identified in spiders and ticks. Cystatins, lipocalins
*Correspondence:[email protected]
DepartmentofPhysicsandChemistry,SchoolofPharmaceuticalSciencesof andpeptidaseS1arefoundonlyinticks[5].
RibeirãoPreto,UniversityofSãoPaulo(USP),AvenidadoCafé,s/n,Ribeirão
Preto,SP14.040-903,Brazil
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(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,
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creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page2of14
The scorpion venom apparatus consists of a gland
connected to a telson sting which is located on the last
segmentofthepost-abdomenoftheanimal(Fig.2).This
is an apparatus of great importance for their survival,
assisting in feeding and self-defense of the scorpion. The
telsonhasavesiclethatcontains apairofglandsrespon-
sible fortheproductionandstorageofthe venom [2].
A scorpion sting is characterized by intense pain and
systemicsymptomsthatusually developrapidly[10].Ac-
Fig.1Venndiagramhighlightingtheproteinfamiliespresentedin cording to clinical manifestations,scorpion envenomings
ticksalivaandscorpion/spidervenoms.Cataboliteactivatorprotein are classified as mild, moderate or severe. The general
(CAP),defensins,hyaluronidase,Kunitz-likepeptides(serineproteinase initial response to a scorpion sting is immediate local
inhibitor),neurotoxins,lectinsandphospholipasearesomeofthe
burning pain, which may be severe. General symptoms
compoundssharedamongthesearthropods
may occur soon after the sting, but may be delayed for
several hours. Therefore, vital functions of patients with
In this context, the study of the structural similarity systemic manifestations should be observed continuously,
among these compounds/toxins identified in the venom/ whileseekingearlytreatmentofthecomplications[11].
saliva of these animals may contribute to a better under- So far, approximately 2,000 species of scorpions have
standingoftheactionmechanisminvolvedinenvenoming been described, distributed worldwide. These arach-
besides providing information about molecules with great nids are classified into seven families: Scorpionidae,
biotechnologicalpotential. Diplocentridae, Chactidae, Vaejovidae, Bothriuridae,
Chaerilidae and Buthidae. The most dangerous species
belong to the family Buthidae, which comprises more
Review than 500 species. In Brazil the scorpions with the
Scorpionvenoms highest medical and scientific interest belong to the
Scorpion envenoming is considered a public health prob- genusTityus [2, 12–15].
lem, especially in tropical countries [7]. Annually, more There are more than ten different Tityus species in
than one million cases of scorpion envenomation are Brazil, among whichTityus stigmurus,Tityus bahiensis
reported worldwide with a fatality risk of around 3 % and Tityus serrulatus are primarily responsible for hu-
[8]. According to the data from the Brazilian Ministry man envenoming. T. serrulatus is considered the most
of Health, 57,933 accidents were recorded in Brazil in dangerous species in the country, responsible for the
2011, of which 91 cases resulted in death [9]. highest number of envenoming accidents [16, 17].
Fig.2Photoofascorpionandschematicrepresentationofscorpions’telson.Morphologyoftheinoculumapparatusofscorpionvenomlocated
onthelastsegmentofthepost-abdomenoftheanimal.Thetelsoncomprisesapairofglandsresponsiblefortheproductionandstorageofthe
venomusedforfeedingandself-defenseofthescorpion
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page3of14
BiochemicalcharacteristicsofthevenomfromTityus Theα-NaScTx retardsthe mechanism ofNavinactivation
Scorpion venoms are a complex mixture of substances and prolongs the repolarization phase of the membrane
thatinclude:inorganicsalts,freeaminoacids,heterocyclic actionpotential[2].Theα-NaScTxcanbesubdividedinto
components, peptides and proteins, mainly enzymes that the following three main groups: (1) classical α-toxins,
are usedby the scorpions for self-defenseand the capture which are highly active only in mammalian Nav channels
of prey [18]. A broad range of bioactive compounds of andpresentpoortoxicityagainstinsects;(2)anti-insectα-
scorpion venoms have already been purified and char- NaScTXs, which are highly active only on insect Nav
acterized. It is estimated that the number of different channels; and (3) α-like toxins, active on both insect and
components present in these venoms is approximately mammalian Nav channels [18]. As shown in Table 1,
100,000, but only 1 % of these molecules have been iso- toxinssuchasTs3isolatedfromT.serrulatus,TbTx5from
lated and characterized [19]. The advent of recombin- T. bahiensis and Tst3 from T. stigmurus are highly con-
ant DNA technology, such as transcriptome analysis, served between the species sharing a high percentage of
allowed the identification of new components; however, identity [29–31]. Those toxins also show high similarity
some of them have not yet been directly purified from withTs5ofT.serrulatusandTb3ofT.bahiensis.TheTs3
the venom. relaxes the human corpus cavernosum in vitro through
Venoms varies compositionally from genus to genus and the release of NO from nitrergic nerves and the elucida-
species to species and may differ in potency, probably due tion of its action mechanism would be useful for the de-
tochangesintheproportionoftheirtoxins,associatedwith velopment of new therapeutic strategies to treat priapism
genetic and environmental variations, such as diet and cli- after scorpion envenomation. Additionally, this is a mol-
mate[20–23].StudieshaveshownthatT.serrulatusvenom ecule that can be used as a model for the development of
is two to three times more toxic than that of T. bahiensis, anewdrugtotreaterectiledysfunction[32].
whichexplainsthevariousstudiesthataimedtoisolateand Another class of toxins that affect Nav channels is the
characterize their toxins [2]. Furthermore, such studies β-neurotoxins (β-NaScTx), which bind to receptor site 4
found variability in venom lethality among T. serrulatus in the extracellular loops that connect transmembrane
specimens,whichsuggeststhatneurotoxins,suchasα-type segmentsS3andS4andtheS1andS2segmentsindomain
neurotoxin, must be the major lethal component in the II [2, 18]. Thus, this class alters the voltage-dependence of
wholevenom[24]. channel activation to more negative potentials to cause an
The major components of scorpion venom are neuro- increased tendency to trigger the spontaneous and the
toxins, which act on ion channels of excitable cells [25]. repetitive potentials of the membrane [2]. Similar to α-
The venom compounds may interact with each other to NaScTx, the β-neurotoxins are subdivided into four
modulate the function of ion channels, which is usually groups according to their pharmacological selectivity
responsible for the known symptoms of envenoming. for insect and mammalian Nav channels: (1) βm, active
Scorpion neurotoxins present a tightly tridimensional- on mammalian Nav channels; (2) βi, selectively active
shaped backbone stabilized by three or four disulfide on insect Nav channels; (3) β-like, for toxins without
bridges. This property avoids their in-vivo degradation, preference between mammalian and insect Nav chan-
therebyincreasingtheirinteractiontimewithionchannels nelsand(4)βα,forthosethatpresentsaprimarystructure
andtheirefficacy[18]. of β-toxins, but with a functional α-effect [14]. The
Four different families of neurotoxins are usually found toxin Ts1, a β-neurotoxin with action on Nav channels,
in scorpion venom: peptides that modulate sodium-, is the most abundant toxin in T. serrulatus venom,
potassium-,chloride- or calcium-gatedchannels[12].The whose activities include inducing macrophage activation
most studied families of venom neurotoxins from Tityus invitro[33,34].
speciesactonsodiumandpotassiumchannels.Thepoorly The neurotoxins that act on voltage-gated K+ channels
known toxins specific for chloride and calcium channels (Kv) can be classified into α, β, γ and κ [35, 36]. There
present variableaminoacid lengths [26].The neurotoxins aretwomaintypesofstructural motifsobservedinthese
present a highly conserved essential three-dimensional peptide classes: (1) the common motif comprised of one
structure comprising an α-helix and three- or four- or two short α-helices connected to a triple-stranded
stranded anti-parallel β-sheets connected by two to antiparallel β-sheet stabilized by three or four disulfide
four disulfide bonds [18, 27, 28]. bonds, denominated CS αβ and (2) the α-helix-loop-
Thescorpiontoxinsthataffectmammalianvoltage-gated helix (CS αα) fold consisting of two short α-helices con-
Na+ channels (Nav) are classified as: α-neurotoxins (α- nected by a β-turn; only the kappa toxins adopt this fold
NaScTx) and β-neurotoxins (β-NaScTx). The α-NaScTx [18, 37–40]. The α-neurotoxins (α-KTx) block the pore
interacts with channel receptor site 3 located in the binding to the external vestibule of the channel and
S3–S4 extracellular loop in domain IV and in the S5– block the ionic conductivity by occlusion of the physical
S6 extracellular linker domain I of Nav channels [2, 18]. pore without affecting the kinetics of channel activation
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page4of14
Table1ExamplesofcompoundsfromTityusscorpionvenoms
Compounds Examples Species MolecularMass ActionMechanism References
(kDa)
Neurotoxins Ts3,Ts5 Tityusserrulatus ~6.0–7.0 ActiononNa+channels 29–32
TbTx5,Tb3 Tityusbahiensis
Tst3 Tityusstigmurus
Ts1 Tityusserrulatus 6890.9 33–34
Ts6,Ts7 Tityusserrulatus ~6.0–7.0 ActiononK+channels 35–40
Tst26 Tityusstigmurus
Tt28 Tityustrivittatus
TdK1 Tityusdiscrepans
Hypotensiveagent Hypotensin Tityusserrulatus 2.75 AgonistoftheB(2)receptor 41
Antimicrobial TsAP1,TsAP2 Tityusserrulatus ~8.4 Unclear 42
peptides
Proteinases Metalloproteinase Tityusserrulatus ~25.0 Lysisofthecellbasementmembrane 43–46
Serineproteinasesa TityusserrulatusTityus – Actiononcoagulationfactors 47
bahiensis
Enzymes Phospholipaseb TityusserrulatusTityus – Hydrolysisofmembrane 48–49
stigmurus phospholipids
Hyaluronidase Tityussp. ~50.0 Catalyzesthehydrolysisofhyaluronan 50
fromtheextracellularmatrix
aIdentifiedinthevenom,butnotpurified
bCompoundfoundonlyinthetranscriptome
[41]. Ts6 and Ts7 from T. serrulatus, Tst26 from T. The other subclasses of neurotoxins that act on Kv
stigmurus, Tt28 from T. trivittatus and TdK1 from T. channels, such as γ and κ, are less studied. However the
discrepans are examples of α-neurotoxins that act on γ-KTxs neurotoxins were described as mainly targeting
Kv channels [35, 42–45]. hERG channels and were found in scorpions of the
In addition to α-KTxs, the venoms of the Buthidae, genusCentruroides, MesobuthusandButhus[18,36].The
Caraboctonidae and Scorpioninae families also contain κ-KTxs neurotoxins show an interaction with voltage-
β-neurotoxins (β-KTxs) [35]. According to the identity gated Kv channels similartoα-KTx toxins, presentingthe
of the sequences, these toxins may be divided into three lysineandaromatic/hydrophobicresidue(functionaldyad)
classes. Class 1 containing the toxins TsTX-Kβ-related thatinteractwiththechannel[18].
peptides, such as TsTx-Kβ,TtrβKTx,TdiβKTx,TstβKTx, The diversity of toxins that target Kv channels with
Tco42.14fromT.serrulatus,T.trivittatus,T.discrepans, high affinity and selectivity provides a large number of
T. stigmurus and T. costatus, respectively. The only molecular structures that can be considered for the
peptide characterized to any extent is TsTx-Kβ from T. development of therapeutic drugs for diseases such as
serrulatus, which is a blocker of Kv1.1 channel with cancer and autoimmune diseases, in which there is an
IC50 values of 96 nM [46]. Class 2 consisting of peptides overexpression of these channels [48]. For example,
homologous to BmTXKβ from Buthus martensii which the HERG channels are associated with cell cycle and
showed an inhibition of the transient outward K+ current proliferation of several cancers; therefore, the use of
(Ito) of rabbit atrial myocytes; some examples of class 2 HERG-specific blockers could inhibit the proliferation
peptides are TdiKIK, TtrKIK, TcoKIK and TstKMK [18]. of tumor cells [18].
Class 3 is formed by the Scorpine-like peptides, also The scorpion venoms are composed of other peptides
known as “orphan” peptides. They possess two structural andproteinssuchashyaluronidases,antimicrobialpeptides,
and functional domains: an N-terminal α-helix (with phospholipases,allergens,hypotensinsandalsoproteinases,
cytolytic and/or anti-microbial activity such as insect such as serine proteinases and metalloproteinases, among
defensins) and a tightly folded C-terminal region with a others.However,someofthesemoleculeswerenotisolated
CS αβ motif, displaying Kv channel blocking activity. from the scorpion venoms and were only identified in
Thescorpinehomologsexhibit strongantimicrobialeffects the venom gland transcriptome.
as well as cytolytic activity against eukaryotic cells and In addition to the neurotoxic effects induced by toxins
possible antimalarical activity [18, 46, 47]. acting on ion channels, a wide variety of actions of the
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page5of14
venomcomponentscanbeobservedsuchashypotensive One of the major goals of the identification and
and antimicrobial effects induced by TsHpt-I and scor- characterization of animal toxins is the possibility of
pine, respectively. TsHpt-I, isolated from T. serrulatus obtaining new therapeutic drugs. A famous example
venom, acts as an agonist of the B(2) receptor and does about scorpion toxins with biotechnological application
not inhibit angiotensin-converting enzyme [49]. As de- is the chlorotoxin isolated from venom of the Israeli
scribed above, the Tityus venom possesses a peptide scorpionLeiurusquinquestriatus,whichwasinitiallydevel-
called scorpine which presents an antimicrobial and oped for the diagnosis and treatment of glioma. Further-
antimalarial activity [47]. Recently, Guo et al. [50] identi- more, this toxin was discovered to be capable of labeling
fied two othersantimicrobialpeptides,TsAP1 andTsAP2, specific cancer cells [63]. Although the biomarker respon-
with broad spectrum antimicrobial and anticancer ac- sible for the binding is still under discussion, it has been
tivities. The antimicrobial peptides are cationic and preliminarily identified as annexin 2A. Recently, the ex-
amphipathic, mostly within 50 amino acid residues, tremely stable iodinated analogue of this toxin—TM601,
were gathered into different groups and their action which presents no immunogenicity and produces no tox-
mechanisms remain unclear [12]. icity in humans—has successfully completed clinical phase
Although the presence of phospholipase was reported II in the treatment of recurrent glioma and was approved
in the transcriptome of T. serrulatus and T. stigmurus, bytheFoodandDrugAdministration(FDA)[63–65].
venoms of T. serrulatus, T. bahiensis and T. stigmurus Thus, given the wealth of components present in scor-
exhibit significant proteolytic but no phospholipase pion venom, it isconcluded that the study of these toxins
activity [51–53]. The venom of these scorpions also is not only a potential source of new drugs, but also a
showed metalloproteinase activity; however, this enzyme source of tools in the elucidation of the physiological sys-
was obtained only from T. serrulatus venom [51, 54–56]. temsandenvenomingpresentedbytheseanimals[66].
Furthermore, enzymes that present gelatinolytic activity,
such as serine proteinases, were detected in T. serrulatus Spidervenoms
and T. bahiensis venoms, but these toxins have not been Spiders possess four pairs of paws and an external skel-
isolatedyet[57]. eton composed of chitin (Fig. 3). The exclusive feature
Hyaluronidase, another important protein present in of these animals is the presence of chelicerae associated
scorpionvenom,isconsidereda“spreading factor”byfa- withthevenomgland,exceptforrarespecies.Thespiders
voring the absorption and spread of venom through the use their venom primarily to paralyze or kill their prey,
tissues of the victim, contributing to local or systemic sometimes for self-defense, which may cause occasional
envenoming [58]. Animals injected with Ts1, the major accidents[67].
toxinfromT.serrulatus,andhyaluronidaseachievedsig- The World Health Organization (WHO) establishes
nificantly higher serum levels of creatine kinase (CK), that only four spider genera contain species capable of
lactate dehydrogenase (LD) and aspartate aminotransfer- causing medically important accidents in humans: Loxos-
ase(AST)inashortertimethanthoseinjectedwithonly celes, Phoneutria, Latrodectus and Atrax [68]. In Brazil,
Ts1 (without hyaluronidase), confirming the characteris- Loxosceles, Phoneutria and Latrodectus are the most rele-
tic of the “spreading factor” of the hyaluronidase. The vant genera and account for a large number of accidents
animals, which received only hyaluronidase, showed CK, inthiscountry[69].
LD and AST levels similar to those of the control group, Spider venom contains a complex mixture of distinct
indicatingnointrinsic toxiceffectof hyaluronidase[59]. compounds[70].Themaincomponentsareneurotoxins,
The advent of transcriptome analysis of the scorpion proteins, peptides, enzymes, free amino acids and inor-
venom gland allowed the determination of several com- ganic salts. Indeed, many toxins isolated from spider
ponents that had not been purified from the venom of venom have been studied in relation to their role in ion
these animals. Transcriptome of several scorpions has channels[71](Table2).
beenperformed,andamongthegenusTityusthetranscrip- Thesecocktailsofsubstancesthatactbydifferentpharma-
tomes of T. stigmurus,T. discrepans,T. costatus Karsch,T. cological mechanisms have been extensively researched
pachyurus,T. obscurus,T. bahiensis and T. serrulatus have seeking to develop new drugs and biotechnological
been reported [52, 53, 60–62]. These analyses found products [72].
transcripts of novel proteins such as phospholipases, The distinct characteristics of venom from each species
metalloproteinases, allergens, proteinases, antimicrobial determineitseffectonhumansintheeventofanaccident.
peptides and anionic peptides. However, the possibility Venom from the genus Loxosceles, or brown spider, has
thatthosetranscriptshadundergonemicroRNA-mediated constituents such as hyaluronidases, metalloproteinases,
degradation during the processing period may explain phospholipasesandotherenzymesthatprovidealocalef-
why some toxins were found only in the transcriptome fectwithdeeplesions,incontrasttothegenusPhoneutria,
andnotinthevenom[53]. whose venom produces neurotoxic activity [73]. The
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page6of14
Fig.3Photoofaspiderandschematicrepresentationofaspider’schelicerae.Cheliceraeareassociatedwithvenomglands,whichare
responsiblefortheproductionandstorageofvenom.Thecheliceraearealsousedtotrapandkilltheprey
Latrodectus genus, or black widow spider, has neuro- and responsible for most accident cases registered in
toxic venom components that act on presynaptic nerves Brazil. Such accidents occur mostly in the south and
ofvertebrates[74]. southeastregionsofthecountry[75,76].
In this review, we focused only on three genera re- Experimentalstudieshaveshownthatthevenomcauses
sponsible for the highest amount of medically important an activation of voltage-dependent sodium channels, and
accidentsin Brazil, Loxosceles, Phoneutria and Latrodectus, a blockade of voltage-dependent potassium and calcium
their principal componentsand respective contributions in channels in muscle fibers and sensory nerve endings in
physio-pharmacologicalstudies. boththemotorandautonomicnervoussystems.Asacon-
sequence,thereisareleaseofneurotransmitters,especially
BiochemicalcharacteristicsofthevenomfromPhoneutria acetylcholine and catecholamines, which explains the fol-
Spiders of the Phoneutria genus are popularly known as lowing symptoms: severe pain at the bite site, sweating,
“armed” due to the attack position they assume in a situ- agitation, salivation and, in severe cases, arrhythmias and
ationofdanger.Whenthesespidersfaceanopponent,they priapism[75,77,78].
raise their front legs and lean on the back legs, presenting This venom is a cocktail consisting of peptides, free
aggressivebehavior[68]. amino acids, histamine, serotonin and serine proteinases
The venom of this genus causes immediate and intense [79, 80]. Furthermore, the Phoneutria nigriventer venom
local pain radiating in the affected limb, but can progress islargelycomposedofneurotoxins.
into complications, especially in children and the elderly, The Phoneutria neurotoxins are similar to those from
such as salivation, sudoresis, hypertension, priapism and scorpion venoms. They present different amino acid se-
even death. These spiders are found in banana plants, quences, but are rich in cysteines forming three or four
palm trees and bromeliads. They are habitually nocturnal disulfidebonds,whichareresponsibleforpeptidestability.
Table2ExamplesofcompoundsfromBrazilianspidervenoms
Compounds Examples Species MolecularMass ActionMechanism References
(kDa)
Neurotoxins PnTx1,PnTx2,PnTx3 Phoneutria ~6.0–9.0 Actonionchannels 72
nigriventer
PnTx4 Phoneutria 5.17 InhibitreversibleNMDAreceptorsininsects 73
nigriventer
α-latrotoxin Latrodectussp. ~130 InfluxofCa2+onpresynapticnerveendings 74
Enzymes PhospholipaseD Loxoscelessp. ~31.0–32.0 Hydrolysisofmembranephospholipids 75
(Sphingomyelinase)
Hyaluronidase Loxoscelessp. – Catalyzesthehydrolysisofhyaluronanfromtheextracellular 76
matrix
Proteinases Metalloproteinase Loxoscelessp. ~29.0 Lysisofthecellbasementmembrane 77
Serinoproteinases Loxoscelessp. ~85–95.0 Actiononcoagulationfactors 78
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page7of14
In this genus, for example, there are three neurotoxins biochemically.Amongthesearehydrolases,hyaluronidase,
lethal to mice, denominated PnTx1, PnTx2 and PnTx3. lipases, metallo—and serineproteinases, peptidases, colla-
The fraction PnTx4 modifies the neuromuscular re- genases,alkalinephosphataseandphospholipaseorsphin-
sponse in insects [75, 79]. gomyelinaseD[93–96].
The PnTx2 fraction is composed of nine different pep- The sphingomyelinases are phospholipases D considered
tides, which are mainly responsible for the overall effect the major components of the venom and are primarily
of the venom. Of these nine peptides, the Tx2-5 and responsible for dermonecrotic lesions. Furthermore, these
Tx2-6 are active in smooth muscle relaxation of the cor- enzymes are related to reactions involving components of
pus cavernosum in rats and rabbits, causing erection the complement system, migration of polymorphonuclear
[81–83]. This fact, along with the discovery that some of leukocytes, platelets aggregation and inflammatory re-
these fractions have insecticidal activity, has drawn the sponse [97].
attention of researchers to the study and characterization Although sphingomyelinase D plays a key role in the
ofthePhoneutriavenom. Loxosceles envenoming and is the major component,
Inaddition, PnTx4was able toinhibit glutamate uptake studieshaveshownthattheclinicalmanifestationsarethe
by rat synaptosomes. The toxin Tx4(5–5), a polypeptide resultofaninteractionbetweenseveralothercomponents
composed of 47 amino acid, displays a potent insecticidal inthevenom[98].
activity. This toxin reversibly inhibited the N-methyl-D- Studies of L. gaucho, L. deserta and L. reclusa venom
aspartate(NMDA)subtypereceptor[84]. demonstrated the presence of metalloproteinases with
A comparison of the proteomes of P. nigriventer, P. gelatinolytic, caseinolytic and fibrinogenolytic activity.
reidyi and P. keyserlingi revealed a large number of These enzymes appear to be involved with the signs and
neurotoxic peptides that act on ion channels, which symptoms of envenoming. Some of these metallopro-
causeparalysisanddeathwheninjectedinmice,aswellas teinases present astacin-like activity. The astacins are
proteinases and peptides with insecticidal activity and zinc-dependent proteinases with such diverse functions
non-toxicpeptides[85]. as hydrolysis, digestion of peptides and degradation of
Spiders contain innumerous peptides with interesting extracellular matrix. These astacin-like metalloproteinases
actions but with a low amount in the venom; for this rea- have been identified in the venom of L. gaucho and L.
son, these components have been synthesized or cloned laeta[93,95,99,100].
andexpressedinbacteriaoryeast.Anexampleisarecom- In addition, two serine proteinases from the same spe-
binantofPnTx-1andPnTx3-4fromPhoneutrianigriventer cies of Loxosceles have been reported to hydrolyze gel-
venom. These studies open new perspectives in drug atin [100, 101]. The authors concluded that the activity
development and research [86, 87]. of serine proteinases complementsother fibrinogenolytic
proteinases in disseminated intravascular coagulation,
triggered by loxoscelic venom [95, 101]. Furthermore,
BiochemicalcharacteristicsofthevenomfromLoxosceles another enzyme that plays a key role in envenoming is
ThedifferentspeciesofthegenusLoxoscelesaredistributed hyaluronidase, which is responsible for the gravitational
globally.TheyarefoundinSouthAmerica,NorthAmerica, effectontheskinthatspreadsthevenom[73,95].
Europe, Africa, Oceania and Asia. They are popularly Toxins from Loxosceles venom have been cloned and
known as brown spiders and comprise more than 30 expressed using cDNA. An example of recombinant pro-
species in South America. In Brazil, the highest incidence teingeneratedbyloxoscelicvenomisLoxoscelesintermedia
ofthesespidersisinthesouthernandsoutheasternregions, recombinant dermonecrotic toxin (LiRecDT), which has
wheretheL.gaucho,L.laetaandL.intermedia speciesare propertiessimilartotheL.intermediavenom,withrespect
found[73,88–90]. toinflammatoryanddermonecroticactivity,andstimulates
A brown spider bite can cause cutaneous or systemic nephrotoxicity in rats [73]. Furthermore, many sphingo-
(or both in some cases) manifestations in the victims. At myelinases have been cloned from the Loxosceles cDNA
leastthreeactions of the loxoscelic venom aredescribed: glands and expressed to obtain larger amounts of this en-
proteolysis with dermonecrosis at the bite site with a zymeandallowstudyofthestructureandfunctionofthese
gravitational lesion; hemolytic action with intravascular toxins[97,98].
hemolysis, which may lead to acute renal failure, and
coagulant activity with thrombocytopenia, hypofibrino-
genemia, prolongation of clotting time and disseminated BiochemicalcharacteristicsofthevenomfromLatrodectus
intravascularcoagulation[91,92]. genus
Brown spider venom is a mixture of toxins composed Worldwide,morethan40speciesofthegenusLatrodectus
of proteins and also low-molecular-weight constituents. are found in tropical and subtropical regions. In Brazil,
Numerous toxins have been identified and characterized onlythreespeciesoccur:L.geometricus,L.mactansandL.
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page8of14
curacaviensis, which inhabit mainly the northeast region three families: Ixodidae, Argasidae and Nuttalliellidae
[102, 103]. However, the presence of another specie, L. [118, 124–126]. There are several genera of ticks, most
mirabilis,wasrecentlydescribedinthesouthernBrazilian importantlyIxodes,Dermacentor,Boophilus,Rhipicephalus,
stateofRioGrandedoSul[104]. Haemaphysalis, Hyalomma and Amblyomma, which
The bites of these spiders, known as black widows, belong to the family Ixodidae [126].
provokeclinicalmanifestationsthatincludepain,hyperten- In Brazil, studies have reported the existence of 55
sion, spasms, “facies latrodectismica”, vomiting, abdominal species, divided into six genera of the family Ixodidae
painandmusclecramping.Inseverecases,thepatientmay (Ixodes, Amblyomma,Haemaphysalis, Anocentor,Rhipice-
present myocardial infarction and compartment syndrome phalus and Boophilus) and four genera of the Argasidae
[102,105]. family (Argas, Ornithodoros, Antricola and Otobius). The
TheLatrodecutsvenomcontainsacocktailofsubstances, Ixodidaefamilyincludesthemostofthespeciesofmedical
but its major component is α-latrotoxin (α-LTX), a neuro- and veterinary importance in Brazil, where the genus
toxinthatactsselectivelyonpresynapticnerveendingsand Amblyomma (the largest genus containing 33 species)
provokes a discharge of neurotransmitters. This toxin is a is the most important in the medical field. The species
protein with high molecular mass (about 130 kDa of ma- Amblyomma cajennense, A. aureolatum and A. cooperi
turetoxin),butshowsnoenzymaticactivity[74,106–110]. stand out in relation to the transmission of spotted
The effects of the LTX seem to be related to the for- fever [127, 128].
mation of pores in the membrane. LTX binds to specific Morphologically, ticks present two fused parts, namely
receptors (named neurexin and latrophilin) which can the capitulum (or gnathosoma) that contains the head
facilitate the insertion of this toxin and subsequent in- andmouthparts,andtheidiosomathatcontainsthelegs,
fluxofCa2+[106,111,112]. digestive tract and reproductive organs (Fig. 4). The ca-
LTXs have targeted insects (latroinsectotoxins), crusta- pitulum consists of three specialized structures: palpus,
ceans (latrocrustatoxin) and mammals. Many of these cheliceraeandahypostome.Nymph andadulttickshave
latrotoxins have been cloned and studied in relation to eightlegswhereaslarvaltickspossesssix[118,124,129].
their structure, maturation and activity. Moreover, these Several diseases can be transmitted during feeding by
toxins can help to elucidate the mechanisms of neuro- ticks, which are obligate hematophagous organisms. Der-
transmitter release and to identify neuronal cell-surface malandepidermaldamage(ruptureoflocalbloodvessels)
receptors[113]. are consequences of the insertion of the tick hypostome
[125–127]. In contrast to the toxins of other arthropods
such as scorpions and spiders, which utilize their toxins
Ticks for protection as well as predation, the advantages of the
Theknown tickborne diseases areofgreat interestinthe ticktoxinsarestillunclearandrequireadditionalresearch
fieldofpublichealth.Ticksarerarelyconsideredvenomous [130, 131]. We will discuss below the main compounds
but some studies provide evidence to the contrary foundinsalivafromBrazilianfamiliesofticks.
[5, 114–116]. Ticks, as vectors of disease transmission to
humans, rank just behind mosquitoes as the most import- Biochemicalcharacteristicsofticksaliva
ant arthropod transmitters of pathogens to several animal Studies performed to evaluate the pharmacological com-
species [117]. Although these diseases have focal features plexity presented by hematophagous arthropods have
on some regions, they have been recognized worldwide. shown that their saliva contains at least one anticlotting,
Virus and bacteria are the main causes of the diseases one vasodilatory and one anti-platelet substance [132].
transmitted by ticks. Among the virus-associated Amongticksalivacomponentsaredescriptionsofenzymes,
diseases, we can cite encephalitis, Crimean-Congo enzyme inhibitors, host protein homologues, amine-
hemorrhagic fever, Omsk hemorrhagic fever, Colorado binding lipocalins, immunoglobulin-binding proteins,
tick fever, Powassan encephalitis, Langat encephalitis and receptoragonist/antagonist,calcium-bindingcomponents,
louping ill encephalitis. Some tickborne diseases associ- cement cytokine components, cytokine expression modu-
ated with bacteria have already been described including lators,non-proteinaceousbioactivecomponentsandother
tularemia, ehrlichiosis (monocytic and granulocity), rick- components related to cardiotoxic and neurotoxic factors
ettsiosis(spottedfever),Lyme borreliosis (Lyme disease) [118,119,127,130,132,133].
as well an infection caused by a protozoan, babesiosis The Amblyomma cajennense is the most studied species
[118–123]. in Brazil. After constructing a cDNA library on this tick, a
Ticksarecosmopolitanandassociatedwithnumerousdis- serine protease Kunitz-type inhibitor was designed. This
eases besides being the most important group of ectopara- newinhibitorknownasAmblyomin-Xwasabletodecrease
sites of wild animals [118, 124]. Today, approximately 899 the number of metastatic events and the tumor mass in a
tick species have been described and distributed among B16F10 murine melanoma model by apoptosis induction
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page9of14
Fig.4Photoofatickandschematicrepresentationofthecapitulum.DorsalandventralmorphologyofthemouthpartofIxodidaefamilyticks.
Onthedorsumitispossibletoobservethecheliceraewhiletheventerdisplaysthehypostome.Thepalpusisobservableonbothsides(dorsum
andventer).Thehypostomeisresponsibleforthedermalandepidermaldamage(ruptureoflocalbloodvessels)duringthetick’sfeeding
[134–136].Moreover,theAmblyomin-Xwasabletoinhibit neurotoxins such as HT-1 (holocyclotoxins) in the
thefactorXafromcoagulationcascade[136].Althoughthis Ixodes holocyclus tick saliva and another still unnamed
species is the most studied in Brazil, most studies have one in the Rhipicephalus evertsi evertsi tick saliva
focused on characterization and therapeutic application [127, 146, 147].
of Amblyomin-X [134–136]. The gene coding of the HT-1 neurotoxin in the saliva
Saliva-enzyme inhibitors have great biotechnological of the tick I. holocyclus showed high homology with the
potential in the medical field. Ornithodorin (Ornitho- gene coding scorpion neurotoxin [114, 146]. The study
doros moubata) and savignin (Ornithodoros savignyi) are of this toxin may help elucidate the potentially fatal tick
examples of potent thrombin inhibitors from tick saliva paralysiscaused bythisarthropod[127,146–157].
[137, 138]. A novel tissue factor pathway inhibitor called The presence of the phospholipase A (PLA ) was ob-
2 2
ixolaris was found through the sialotranscriptome ana- served in saliva from Amblyomma americanum. This en-
lysis of I. scapularis [139, 140]. Among the inhibitors of zyme is secreted in the tick-host interface, and probably
factor Xa, Salp14 is the main prototype identified in I. playsanimportantroleduringprolongedtickfeeding.The
scapularis saliva, whereas tick anticoagulant peptide (TAP) PLA does not contribute to the anticoagulant activities
2
is the main inhibitor of factor Xa from Ornithodoros but is associated with hemolytic activity observed during
moubata [141–144]. Variegin isolated from Amblyomma feeding[158,159].
variegatumsaliva is one of the smallest thrombin inhib- Some lectins were characterized in the ticks O. mou-
itors (3.6 kDa) identified in nature. This inhibitor binds bata (Dorin M and OMFREP) and I. ricinus (ixoderin
to thrombin with strong affinity and is considered an A and ixoderin B). Lectins play roles in the innate im-
excellent model for the development of new inhibitors munity of ticks whereas that of R. microplus induces
of this class [145]. immunosuppression in mice [5, 160–162].
In contrast to the scorpions, few neurotoxins were Anantimicrobialproteinwasidentifiedinthehemolymph
found in tick saliva to date. Some studies described of the tick Amblyomma hebraeum and denominated
Cordeiroetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2015) 21:24 Page10of14
hebraein (11 kDa). Native hebraein and its recombinant showing antimicrobial activity against all standard
form, named hebraeinsin, revealed antimicrobial strains [168].
activitiesagainstthegram-positiveandgram-negativebac- Defensins are small proteins present in vertebrates, in-
teria (S. aureus and E. coli, respectively) and the fungus vertebrates and plants and are responsible for their
Candida glabrata [163]. In another study, two non- defense against severalmicroorganisms. Twoisoformsof
cationic defensin-like antimicrobial peptides, designated the defensin gene, denominated def1 and def2, were
Amblyomma defensin peptide 1 and Amblyomma defen- found in saliva of Ixodes ricinus ticks; synthetic peptides
sin peptide 2, were found in the Amblyomma hebraeum from these defensins were tested against bacteria and
tick saliva [164]. The Amblyomma defensin peptide 2 yeast [169]. These defensins showed an antimicrobial
showedantimicrobialactivityagainstE.coliandS.aureus. activity against gram-positive bacteria, but were not ef-
Ixosin, another antimicrobial peptide, was isolated from fectiveagainst gram-negativeonesoryeast[169].Struc-
salivaryglandsofthetickIxodessinensis.Thispeptidehas turally, these defensins contain six cysteine residues
23 amino acids (without cysteine) and showed antimicro- and present as their main action mechanism cell mem-
bialactivityagainstE.coli,S.aureusandC.albicans[165]. brane lysis by a formation of channels [169]. With the
Ixosin-B was purified and cloned from salivary glands of increasing number of microorganisms resistant to con-
the Ixodes sinensis and showed antimicrobial activity ventional antibiotics, the saliva of ticks is becoming an
againstE.coli,S.aureusandC.albicans[166].ISAMP,an important source for the discovery of new compounds
antimicrobial peptide from Ixodes scapularis saliva, has a to treat several diseases.
molecular weight of 5.3 kDa and exhibited antimicrobial
activity against gram-negative and gram-positive bacteria.
Additionally, it showed insignificant hemolytic action Conclusions
on rabbit red blood cells, suggesting that it is a safe In this review we have highlighted the main biologically
antimicrobial peptide for possible use on mammals [167]. active componentspresentinscorpionandspider venoms,
Table 3 summarizes the major components found in the as well as tick saliva, which are of great importance in
ticksaliva. the medical field in Brazil. We have also shown that
After the identification of molecules with important thestudyofarachnidvenomsandsalivaprovidesnumerous
pharmacological actions from natural sources, another compounds with great biotechnological potential. The bio-
possible alternative to obtain peptides is chemical chemical characterization of these compounds, combined
synthesis. Zheng et al. [168] synthetized a defensin- with the advent of molecular biology techniques, enables
like antimicrobial peptide obtained from a cDNA li- the development of new biotechnological products with
brary of the male accessory glands of Haemaphysalis relevantapplications.Additionally,thisstudyallowstheun-
longicornis.This peptide, based on the predicted mature derstanding of the physiological processes involved in the
portion of HlMS-defensin, was tested against a variety envenomings and diseases transmitted by ticks, thereby fa-
of gram-positive and gram-negative bacteria and fungi, cilitatingtheobtainmentofamoreeffectivetherapy.
Table3Examplesofcompoundsfromticksaliva
Compounds Examples Species MolecularMass MechanismofAction References
(kDa)a
Enzyme Amblyomin-Xb Amblyomma 15.0 FactorXaInhibition/inductionofapoptosisin 134–136
Inhibitors cajennense tumorcells
Savignin Ornithodorossavignyi 14.1 Thrombininhibitor 137–138
Ixolaris Ixodesscapularis 18.4 Tissuefactorpathwayinhibitor 139–140
Variegin Amblyomma 3.6 Thrombininhibitor 145
variegatum
Neurotoxin HT-1 Ixodesholocyclus 7.8 Unclear 114,146–
(Holocyclotoxins) 148
Enzyme PhospholipaseA Amblyomma 55.7±1.3 Hydrolysisofmembranephospholipids 158–159
2
americanum
Proteins Hebraein Amblyomma 11.0 Unclear 163
hebraeum
Ixosin Ixodessinensis 8.8 Unclear 165
ISAMP Ixodesscapularis 5.3 Unclear 167
aDataobtainedfromreferencesanduniprot.org
bCompoundfoundonlyinthetranscriptome