Highlights in the knowledge of brown spider toxins

12 Pages·2017·0.67 MB·English

Chaves-Moreiraetal.JournalofVenomousAnimalsandToxins
includingTropicalDiseases (2017) 23:6
DOI10.1186/s40409-017-0097-8
REVIEW Open Access
Highlights in the knowledge of brown
spider toxins
Daniele Chaves-Moreira1, Andrea Senff-Ribeiro1, Ana Carolina Martins Wille1,2, Luiza Helena Gremski1,
Olga Meiri Chaim1 and Silvio Sanches Veiga1*
Abstract
Brown spiders are venomous arthropods that use their venom for predation and defense.In humans, bites of
theseanimals provoke injuriesincluding dermonecrosis withgravitationalspread of lesions, hematological
abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are
called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins
(4–40 kDa). Characterization of the venom confirmedthe presence of threehighlyexpressed protein classes:
phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels
ofexpression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins),
hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D
family (also known as the dermonecrotic toxin) is themost studied class of brown spider toxins. This class of toxins
single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure.
The functional role of thehyaluronidase toxin as a spreading factor inloxoscelism has also been demonstrated.
However, thebiological characterization of other toxins remains unclear and the mechanism by which Loxosceles
toxins exert theirnoxious effects is yet to be fully elucidated.The aim ofthis review is to provide an insight into
brown spider venom toxins and toxicology,including a description of historical data already available inthe
literature. In this review article, the identification processes of novelLoxosceles toxins by molecular biology and
proteomic approaches, their biological characterization and structural description based onx-ray crystallography
and putative biotechnological uses are described along with thefuture perspectives in this field.
Keywords: Brown spider, Loxosceles, Venom, Toxins, Loxoscelism, Phospholipase-D, Metalloprotease, Insecticidal
peptides, Serineprotease, Hyaluronidase
Background bite[2].Brownspidersarecommonlyfoundinworkplaces
Since the brown spider, an arachnid of the genus Loxos- with secluded, dry, sheltered areas such as underneath
celes(Araneae, Sicariidae),can befound worldwide, ithas structures, logs, or in piles of rocks or leaves. The brown
different common names depending on the region it is spiderisalsoadaptedtoliveindoors,theycanbefoundin
found, including brown recluse, violin spider and fiddle- darkclosets,insideshoes,orattics[6,7].Eventhoughthe
back spider [1–4]. The Loxosceles genus was described by genus Loxosceles comprises approximately 130 species
Heineken and Lowe in 1832 [3, 5]. These spiders are and all of them are probably capable of producing clinic-
brown in color with a characteristic dark violin-shaped allysignificantbites, thespeciesresponsiblefor envenom-
mark on cephalothorax and have six equal sized eyes dis- ation in the United States are Loxosceles reclusa,
tributed in semi-circular fashion [6, 7]. The individuals LoxoscelesdesertaandLoxoscelesarizonica.InBrazil,Lox-
present sexual dimorphism, the females usually have oscelesintermedia, Loxoscelesgauchoand Loxosceles laeta
larger abdomens and can inject more venom when they are considered tobe themostimportant spidersfrom the
medicalpointof view[4,8–11].Spiderenvenomationisa
seriouspublichealththreatinBrazilduetothenumberof
*Correspondence:[email protected]
1DepartmentofCellBiology,FederalUniversityofParaná(UFPR),Curitiba,PR, cases recorded annually [12]. In 2015, 26,298 spider bites
Brazil were recorded in Brazil, including 30 fatal cases [13].
Fulllistofauthorinformationisavailableattheendofthearticle
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InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
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Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page2of12
SpidersoftheLoxoscelesgenusareoneofthefourgroups inthreespeciesofLoxoscelesdescribingnewantigenicre-
of spiders that produce venoms that can cause significant gions important to induce neutralizing antibodies. These
clinical manifestations in human or even fatalities follow- synthetic peptides where used to develop an in vitro
ingenvenomation[14].Theconditionthatcommonlyap- method to evaluate the neutralizing potency of horse
pearsafteraccidentsinvolvingLoxoscelesspidersisknown hyperimmunesera(anti-Loxoscelessera)[26].
as loxoscelism and is characterized by several reactions. Epitopes of a recombinant dermonecrotic toxin from
Althoughmostbitesarebenignandlocal,systemicsymp- L. intermedia venom were also used to construct a
toms can emerge [6]. Local reactions include dark blue- chimeric protein called rCpLi. In this study, the authors
violet colored necrotic wounds with gravitational spread, demonstrate that horses immunized with three initial
whicheventuallybecomeindurated,andultimatelyleadto doses of crude venom followed by nine doses of rCpLi
scarring [2, 8]. In nearly half of the cases, cutaneous le- generate antibodies with the same reactivity as those
sionsareassociatedwithnon-specificsystemicsymptoms, produced following immunizationexclusivelywith whole
including fever, fatigue, headache, vomiting, pruritus and venom. They argue that the use of this new generation
rash [8, 11, 15]. Systemic loxoscelism is a less frequent of antivenoms will reduce the suffering of horses and
complication (occurring in up to 13% of the cases) that devastationofarachnidfauna[27].
usually affects children, and leads to manifestations such Diagnosis of loxoscelism is difficult and usually
as renal failure and hematological disturbances, i.e., dis- presumptive. It is often made through evolution of the
seminated intravascular coagulation and intravascular clinical picture and epidemiological information, since
hemolysis[7,11,16,17].Thefirstclinicalcasesofloxosce- few patients bring the animal for its identification [23].
lism were published in the literature describing both Recently, an experimental study developed a recombin-
cutaneousandcutaneous-visceralreactions[18–20]. ant immunotracer based on a monoclonal antibody that
The treatment for loxoscelism includes mainly anti- reacts with L. intermedia venom components of 32–
venom, corticosteroids and dapsone. However, there are 35 kDa and neutralizes the dermonecrotic activity of the
noclinicaltrialstosubstantiateanymethod.Inaddition,it venom. This antibody was re-engineered into a colori-
isdifficulttoevaluatetheefficacyofthetreatmentbecause metric bifunctional protein (antibody fragment fused to
of the diverse forms of cutaneous lesions and often late alkaline phosphatase) that proved to be efficient in two
diagnosis. While systemic corticosteroids are widely used stated immunoassays. This immunotracer could become
inBrazil–eitheraloneorassociatedwiththeantivenom– a valuable tool to develop immunoassays that may facili-
dapsone is frequently used in the USA, although there is tate a rapid and reliable diagnostic of loxoscelism [28].
noconsensusontheefficacyofthesetreatments[21]. As the cases of loxoscelism became noteworthy, Loxos-
Indications for antivenom therapy depend mainly on the celes spider venoms started to be investigated and bio-
time of progression – the earlier the therapy is performed logically and biochemically characterized. This review is
thegreatertheefficacy.Thiswascorroboratedbyanexperi- focused ondifferentaspectsof venom components,such
mental study that showed that necrotic injuries in rabbits as studies in toxinology employing 'omics' strategies and
were about 90% smaller compared with the control when recombinant toxins. The following sections present a
theantivenomwasadministeredupto6h,whilethereduc- historical perspective of the accumulated knowledge re-
tioninthelesiondroppedto30%whentheantivenomwas gardingthebrown spider venom.
administeredupto48hafterthebite[22].Healthprotocols
inBrazil, PeruandArgentina advisetheuseof intravenous History of the brown spider venom toxinology
antivenom in cases of cutaneous or cutaneous-hemolytic Beginningofthevenomstudy
forms of loxoscelism – when hemolysis is present the Loxosceles spider venoms have been studied for over
antivenomisindicatedeven48hafterthebite[21]. 60 years (Fig. 1). Different scientific research groups
However, antivenom therapy may lead to anaphylactic across the world started the process of venom extraction
reactions. A clinical study showed that almost one third and characterization, motivated by the several reports of
ofthe patients who receivedantivenom manifestedsome human loxoscelism cases. Earlier, due to technical limi-
type of early anaphylactic reaction [23]. Experimental tations, the studies were based only on the in vitro and
studies demonstrate some efforts in this direction by in vivo experimental observations. These observations
developing alternative means to elicit a protective im- yielded insight into the pathophysiology of cutaneous
mune response against the noxious effects of dermone- arachnoidism. The first experimental study of loxosce-
crotic toxins, such as using an immunogenic synthetic lism available in the literature was described by
peptide or a neutralizing monoclonal antibody that Macchiavello in 1947 [29]. That report described the
protectrabbitsmainlyagainstdermonecrotictoxinactivity stages of dermonecrosis in guinea pigs after spontaneous
[24,25].Inthiscontext,anotherstudydeepenedthisissue bite by Loxosceles laeta. The first studied venom of
whenitidentifiedpeptideepitopesofrepresentativetoxins brown spider was extracted from Loxosceles laeta and,
Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page3of12
Fig.1Majorhistoricalevolutionontheknowledgeonbrownspidervenom.MainpublicationsintoxinologyonLoxoscelesspiders
afterwards, from Loxosceles reclusa [29–32]. Since then, spiders were determined by SDS-PAGE electrophoresis
several studies on Loxosceles venoms and toxins were and were analyzed [35]. These Loxosceles venoms pre-
published and this subject attracted the attention of sented a similar protein profile and were enriched in low
severalscientistsandresearchgroups(Fig. 2). molecular mass protein molecules. Molecular mass
analysis revealed three main groups of proteins with
Separationofthevenomcomponents different molecular masses −30-40 kDa, 20–30 kDa and
During the end 1960’s and early 1970’s extraction of 2–10 kDa [35, 36]. The toxicity profiles of Loxosceles
brown spider venom started along with isolation of indi- venoms were similar between female and male speci-
vidual components [33, 34]. According to the observa- mens, and between distinct species, such as L. laeta, L.
tions of Morgan in 1969 [34], the clear, highly viscous reclusa, L. intermedia, L. adelaida, L. similis and L.
venom extracted from an adult female spider contained gaucho. Partial purification of the venom toxins by
on average 50 μg of protein. Moreover, the venom ex- sephadex gel filtration revealed three major fractions;
tracted from eight males and eight females of L. reclusa fraction A, with hyaluronidase activity; fraction B, re-
sponsible for major dermonecrotic activity; and fraction
C, devoid of dermonecrotic activity [33, 34, 37–40]. Fur-
thermore, protease, esterase, and alkaline phosphatase
activitieswere reportedinLoxoscelesvenom [35–39,41].
Demonstrationofthebiologicaleffectsofthevenom
The number of investigations, regarding the toxicity and
pathophysiologicaleffectsofLoxoscelesvenom, increased
together with the development of scientific techniques.
The use of preparative gel electrophoresis and gel
filtration provided tools for investigation of each protein
fraction from brown spider venom [42–44]. Cation-
exchange chromatography at pH 4.0 purified the toxin
fraction responsible for lethality in mice, induction of
Fig.2NumberofscientificpublicationsonLoxoscelesduringthelast necrosis in rabbits, calcium-dependent hemolysis of
60years.Graphswerepreparedusingthenumberofarticles human erythrocytes, and a decrease in the calcium-
retrievedinPubMed(http://www.ncbi.nlm.nih.gov/pubmed)using
induced coagulation time of human plasma [45]. Indeed,
‘Loxosceles’inallfieldsasfilterofsearch,inJuly2016
a fraction of the L. reclusa venom has also shown to
Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page4of12
produce hematological effects in albino mice [46, 47]. inflammatory reaction induced by L. gaucho venom [55].
Similar effects were observed with L. laeta venom in Plateletswerealsoshowntohavearoleininflammation,
rabbits.Therewerestudiesthatdemonstrated abnormal- besides being also involved in local thrombotic disorders
ities in the blood coagulation process, including induced by Loxosceles venom. L. gaucho venom induced
alterations in thromboplastin time, prothrombin time, aggregation of platelets, activated adhesion to collagen
platelet count and fibrinogen-fibrin degradation [48]. and increased the expression of ligand-induced binding
Moreover, a low molecular mass peptide fraction of L. site 1 (LIBS1) and P-selectin, demonstrating the pivotal
reclusavenom wasshowntocontain lethal andneuroac- role of platelets in the development of dermonecrosis
tive componentstoinsects[49]. [56]. On the other hand, another study showed that the
Despite the significance of studying protein fractions platelets have a role in minimizing the hemorrhagic
of brown spider venom, some recent and relevant stud- phenomena and the inflammatory and wound-healing
ies focus on the mechanics of action of whole venom processes, since platelet depleted rabbits showed more
even though sometimes making a parallel with specific severe reactions after Loxosceles venom application [57].
toxins. Systemic loxoscelism, for example, was the sub- Despite all these studies demonstrating important mech-
jectoftwostudiesthatfocused onrenalandcardiactox- anisms by which Loxosceles venom lead to the main
icity [50, 51]. It was observed that L. gaucho venom injuries observed after envenomation, it is known that
caused early acute kidney injury in rats probably due to the venom is a mixture of several hundred biologically
an impaired renal flow and systemic rhabdomyolysis. active compounds that act synergistically. Thus, the
The authors also showed that renal damage is independ- detailed mechanism of action of Loxosceles venoms
ent of a dermonecrotic injury or blood pressure changes remains unknown andisstill object ofstudy.
[51]. Moreover, cardiotoxic effects of L. intermedia
venom were studied in mice and results demonstrated
that venom antigens were detected in the heart and that Biochemicalcharacterizationofthevenomcomponents
the venom induced an impairment in the heart function. Barbaro et al. [58], in 1992, used gel filtration to identify
The authors argue that these cardiotoxic effects could a 35-kDa fraction of L. gaucho venom. This fraction was
play a role in the symptoms of systemic loxoscelism, and found to have dermonecrotic, immunogenic and life-
that loxtox proteins are important to develop the heart threatening activities; it was also the first antigen to be
dysfunctioninenvenomed mice[50]. detected by antibodies during the course of
Aiming to investigate the vascular disorders often as- immunization. This 35-kDa fraction purified from L.
sociated with venom exposure, Nowatzki et al. [52, 53] intermedia venom was found to be able to be incorpo-
analyzed the effects of L. intermedia venom on endothe- rated into human erythrocytes membranes and render
lial cells in culture in two different studies. They showed them susceptible to the alternative pathway of comple-
that the venom primarily induces specific changes to ment. A functional analysis of this venom fraction
cellular adhesion followed by cell retraction, detachment indicated the presence of sphingomyelinase activity and
and, finally, drives an apoptotic mechanism known as thatitwascapable ofinducingalltheinvivoeffectsseen
anoikis. These effects may lead to capillary vessel fragil- with whole spider venom, including C-dependent
ity and facilitate the observed hemorrhagic outcome hemolysisanddermonecrosis [59].
[53]. Moreover, endothelial cell endocytosed the toxins Protease activities were also found in brown spider
of L. intermedia venom but, as no lysosomal damage venoms, with distinct molecular mass profiles and sub-
was observed, the authors argue that deleterious effects strate preferences [60, 61]. Based on the enzymatic fea-
on these cells are not caused by internalization of toxins tures, they were classified as metalloproteases and
[52]. Cultured keratinocytes exposed to L. laeta venom serinoproteases. Two brown spider metalloproteases
increased the expression/secretion of MMP2, MMP9 were identified, namely loxolysin A (20 kDa), with activ-
and MMP7, which was associated with cell death. These ity on fibronectin and fibrinogen, and loxolysin B
effects upon keratinocytes are likely to contribute to the (30 kDa), with gelatinolytic activities [60]. Regarding the
pathologyofcutaneousloxoscelism[54]. presence of metalloproteases in Loxosceles venom, two
The release of inflammatory mediators after inocula- proteases were also found in L. rufescens venom, a
tion of L. gaucho venom in mice footpads was investi- 23-kDa fibrogenolytic protease and a 27-kDa gelatinoly-
gated and results showed a marked PGE release tic protease. Their activities were inhibited by 1,10-phe-
2
associated with an increase of interleukin-6 (IL-6), nantroline, confirming the metalloprotease characteristic
monocyte chemoattractant protein-1 (MCP-1) and kera- of the protease [62, 63]. The degradation of fibrinogen
tinocyte chemoattractant (KC). Edema and leukocyte was reported to occur due to different Loxosceles
migration to the site of inoculation was also observed, venoms; again, inhibition of degradation by 1,10-phe-
thus suggesting that these mediators contribute to the nantrolinewasalsoreported[64,65].
Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page5of12
Serineproteases were detected in L. intermedia venom
by zymographic assays showing two gelatinolytic signals
with high molecular masses (85 kDa and 95 kDa) [61].
The biochemical nature of these proteases was charac-
terized by total inhibition of gelatin hydrolysis using
distinct serineprotease inhibitors such as aprotinin,
benzamidine, leupeptin, PMSF, and soybean-trypsin
inhibitor[61].
Later on, the first description of peptides from the in-
hibitor cystine knot family (ICK) in Loxosceles venoms
was published by de Castro et al. [66]. These small pep- Fig.3Loxoscelesrecombinanttoxins.Graphshowsthepercentage
ofrecombinantLoxoscelestoxinsdescribedintheliteratureclassified
tides isolated from the venom of L. intermedia demon-
byclassoftoxins.Ineachtypeoftoxin,thenumberofrecombinant
strated insecticidal activities, and were named LiTx1,
isoformsisavailable
LiTx2, and LiTx3. These components are polypeptides
with molecular masses ranging from 5.6 to 7.9 kDa, pre-
senting insecticidal activities against highly destructive
pests such as Spodoptera frugiperda and Spodoptera D toxins in L. intermedia crude venom [73]. Indeed, at
cosmioides. Further analysis of the sequences pointed to least 11 phospholipase-D isoforms were identified in the
the presence of possible post-translational modification venomproteomeofL.gaucho,corroboratingthepresence
regionsinthesequencesofLiTx1-3,suchasN-myristoy- of several different dermonecrotic toxins in the Brown
lation, amidation, and casein kinase II phosphorylation spidervenom[74].
sites.Basedonthesequencesofthesetoxins,theauthors Using RNA sequencing, 23 complete sequences of
proposed that LiTx-3 may act on NaV (voltage-gated so- phospholipase-D proteins (PLD) were found in L. similis
dium) channels and that LiTx-2 and 3 may act on NaV venom gland and classified as loxtox proteins [71, 75].
orCaV(voltage-sensitive calcium)channels[66]. Seven different isoforms of phospholipase-D were gener-
ated as recombinant proteins, namely LiRecDT (Loxos-
Omicsandrecombinantvenomcomponents celes intermedia recombinant dermonecrotic toxin) and
Molecular biology techniques were essential for under- these enzymes have also been classified as members of
standing the toxicology of Loxosceles venoms. The the LoxTox family [75–80]. Several other isoforms have
amount of venom (volume and protein) that can be ex- also been identified in the venoms of Loxosceles reclusa,
tracted from each spider is small, hampering the process Loxosceles laeta, Loxosceles arizonica, Loxosceles similis,
of isolation of single native toxins. The first toxin to be Loxosceles boneti, and Loxosceles deserta [81–89]. Stud-
cloned and studied in the recombinant form was a ies comparing recombinant isoforms with distinct
sphingomyelinase-D from L. laeta venom in 2002 by capacities for degrading substrates have demonstrated
Fernandes-Pedrosa et al. [67]. In the same year, differences in the intensity of the effects of these
Kalapothakis et al. [68] cloned and expressed a proteins[90].
functional sphingomyelinase-D from L. intermedia Most enzyme isoforms from the Loxosceles genus have
spider venom and demonstrated its immunological been heterologously produced in prokaryotic systems
properties. Acharacterization ofaphospholipaseD from using E. coli, and large amounts of the soluble and enzy-
L.gaucho wasalsoreported[69].Nowadays,there are24 matically active forms of these proteins are easily
reports of recombinant toxins from Loxosceles in the obtained. The knowledge of PLD sequences allowed the
literature(Fig. 3). development of promising tools, such as a recombinant
The L. laeta venom gland transcriptome analysis re- chimeric protein immunogen expressing epitopes of a
vealedthat16.4%ofthetotaltoxin-encodingESTsbelong dermonecrotic toxin from L. intermedia venom, which
to sphingomyelinases-D [70]. Recently it was found that was atoxic and capable of inducing dermonecrotic and
15% of the whole L. similis venom gland transcriptome hemorrhagic protection [91]. Brown spider phospholi-
corresponds to phospholipase-D transcripts [71]. More- pases D catalyzes the hydrolysis of phospholipids, such
over, the L. intermedia transcriptome analysis revealed as sphingomyelin (SM), at a terminal phosphodiester
morethan20.2%ofalltoxin-encodingESTsfromL.inter- bond to release choline and produce ceramide 1-phos-
media venom gland correspond to phospholipases D and phate(C1P)[73,90,92].Thecatalysismediatedbyphos-
representsasignificantproportionofthetoxinspresentin pholipases D in the presence of Mg+2-cofactor leads to
the brown spider venom [72]. Corroborating these find- hydrolysis of lysophosphatydilcholine (LPC) and release
ings,two-dimensionalgelelectrophoresisdemonstratedat of lysophosphatidic acid (LPA) [81, 92, 93]. It seems that
least 25 spots immunologically related to phospholipases the production of these bioactive metabolites can
Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page6of12
promote upregulation of proinflammatory molecules found were transcripts similar to LiTx-3 (32%), LiTx-2
and exert deleterious effects after exposure to brown (11.4%),LiTx-1(6.2%),andLiTx-4(3.7%) [72].
spiderphospholipasesD[90,92,94–99]. In fact, it was reported that the cloning and produc-
Alternatively, some authors stand up for that tion ofarecombinantpeptide from L.intermediavenom
phospholipase-D toxins (testing recombinant toxins and had a great similarity with the ICK family of peptides,
whole venoms) exclusively catalyze transphosphatidyla- especially LiTx-3 [105]. The recombinant peptide,
tion rather than hydrolysis, forming cyclic phosphate named U2-sicaritoxin-Li1b (U2- SCRTX-Li1b), was used
products from both major substrates – SM and LPC as a tool that enabled the demonstration of an antigenic
[100].Itwasalsoshownthatasphingomyelinase-Dfrom cross-reactivity of antisera raised against crude venom of
Loxosceles arizonica (Laz-SMase D) is a potent insecti- L. intermedia, L. gaucho, and L. laeta with U2-SCRTX-
cidaltoxin [101]. Li1b. This cross-reactivity corroborates the presence of
The first metalloprotease, cloned and expressed from ICK-like toxin members in these Loxosceles venoms,
the cDNA library, was extracted from Loxosceles inter- thus strengthening the idea that this toxin family is
media venom gland, and was characterized as an astacin- widespreadthroughoutthegenus[105,106].
like protease. This astacin metalloprotease presented a
catalytic domain of 18 amino acids – HEXXHXXGXX- StructuralanalysisofLoxoscelestoxins
HEXXRXDR – and a conserved methionine involved ina The first structural study on Loxosceles toxins was per-
sequence turn, met-turn, and zinc-dependent activity formed by Zela et al. in 2004 [107], in which the
(MXY) [102]. The recombinant Loxosceles intermedia crystallization and preliminary crystallographic analysis
astacin-like protease (LALP) promoted endothelial cell of a sphingomyelinase-D from L. laeta spider venom
cultures de-adhesion, in vitro degradation of fibronectin, were performed. Crystal structure of LiRecDT1 from L.
fibrinogen,andgelatin[63].Astacinproteasescomprisesa intermediawaspublishedbydeGiuseppe etal.[108],in-
family of toxins in L. intermedia venom, two other iso- dicating that this toxin contained an additional disulfide
forms, named LALP2 and LALP3 were also described bond in the toxin structure catalytic loop compared with
[103].Besides,astacinswereidentifiedinL.laeta(LALP4) the previously described phospholipase-D from L. laeta
and L. gaucho (LALP5) venoms, suggesting the existence [109, 110]. The phospholipase-D from L. gaucho was
of an interspecies toxin family and revealing the import- also crystallized by Ullah et al. [111] in 2014 and the
ance of these metalloproteases as components of Loxos- structure was shown to be very similar to the
celesvenom[104]. phospholipase-D from L.intermedia[112].
Interestingly, when transcriptome complete analysis of The structural details of the molecules reflect the dis-
L.intermediaandL.laetavenomglandswereperformed tinct enzymatic behaviors of the venom from different
these studies revealed that astacin metalloproteases are species. Phospholipase-D with different structures could
included among the high expressed toxins [70, 72]. In L. have different substrate affinities or enzymatic activities;
intermedia venom gland, astacin transcripts comprise therefore, these differences could explain the clinical
more than 22% of the toxin-encoding transcripts and symptomsorseverityobservedatthelocalbitesiteorthe
represent 8% of the total transcripts in L. laeta venom systemic effects during envenomation by different species
gland [70, 72]. Loxosceles proteases (metalloproteases of the Loxosceles genus. In addition, structural analysis of
and serineproteases) account for 23.1% of the total the catalytic site provided important insights into the
toxin-encoding transcripts in L. intermedia venom enzymaticactivitiesofeachisoform[108,110,112].
gland, second only to the insecticidal peptide sequences Comparisons of the amino acid sequences of spider
that comprise the majority of expressed toxins. In venom PLDs indicate that these proteins contain either
addition, the analysis of proteases in the L. intermedia, 284 or 285 amino acids and display a significant degree
L. laeta, and L. gaucho venoms using two dimensional of homology, mainly with regard to the catalytic import-
western blotting and zymogram, demonstrated a great ant residues [85]. The single polypeptide chain folds to
content of active proteases among the three analyzed form a distorted TIM-barrel, which is lined with eight
venoms, corroborating the high mRNA expression parallelβ-strandsinternally linked byshort flexible loops
reportedonthetranscriptome analysis [104]. to eight α-helices that form the outer surface of the
Regarding the ICK peptides in Loxosceles venom, tran- barrel [110].The catalytic loopisstabilized byadisulfide
scriptome analysis of L. intermedia venom gland found bridge (Cys51 and Cys57) in the L. laeta and with a
that ICK peptides comprise 55.6% of toxin-encoding second disulfide bridge (Cys53 and Cys201) in the L.
messengers [72]. Previously described ICK peptides intermedia, which links the catalytic loop to the flexible
(LiTx1-3) were found and a novel ICK peptide from L. loop to significantly reduce the flexibility of the latter
intermedia, LiTx-4, was identified, and later described loop [108–110]. The catalytic site, Mg2+ binding site,
by the authors. The most abundant toxin transcripts and the substrate binding site are located in a shallow
Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page7of12
depression that contain His12, Glu32, Asp34, Asp91, recombinant molecules [122, 123]. New isoforms of the
His47, Lys93, Tyr228, and Trp230, which are very con- previously described and studied toxins served as tools
served in Loxosceles PLD isoforms [108, 110]. The that strengthened the knowledge concerning venom
importance of theses residues was confirmed by site- actions andloxoscelism[76,78–80,102, 104,124].
directed mutagenesis and the X-ray structural studies The identification of hyaluronidase activity in Loxos-
indicating involvement of the two histidines (His12 and celes venoms comes from a study of L. reclusa venom,
His47) in close proximity to the magnesium coordin- which demonstrated hyaluronidase activity upon hyalur-
ation (Glu32, Asp34, and Asp91) that promote the acid- onic acid (HA) and condroitin-sulphate (CS) types A, B,
base catalytic mechanism. Furthermore, the residues and C [39]. The medically important venoms from five
Lys93,Tyr228, and Trp230 were shown to be important Loxosceles species in the US (L. deserta, L. gaucho, L.
for recognition and stabilization of the substrate intermedia, L. laeta, and L. reclusa) contain a 44-kDa
(phospholipid) duringthe catalytic process[113,114]. hyaluronidase, which is able to degrade HA detected by
Several mutants of PLDs were studied recently bring- zymogram assays [65]. All these identifications of Loxos-
ing light in the understanding of the catalytic and celes hyaluronidases suggest the biological conservation
recognition sites [114, 115]. However, the variety of mo- and significance of these enzymes [65]. Two hyaluroni-
lecular mechanisms triggered by Loxosceles dase molecules of 41 and 43 kDa were characterized as
phospholipase-D toxins and their lipid metabolites pH-dependent endo-β-N-acetyl-d-hexosaminidases
should be further investigated as a complex event hydrolases in L. intermediavenom [124]. These enzymes
dependent on the types of cells involved, the abundance, were able to degrade HA and CS in vitro and HA in
and availability of the lipid substrate, and intracellular rabbit skin[124].
and extracellular signaling cascades [97, 116]. For now, Corroborating the identification of hyaluronidase ac-
it is demonstrated that phospholipases D from different tivity, a proteomic study also described the presence of
Loxosceles species have the ability to reproduce many ef- hyaluronidases in Loxosceles venoms [125]. Loxosceles
fects of the cutaneous and cutaneous-visceral loxosce- hyaluronidase shows high activity, requiring few micro-
lism. They are described as being responsible for several grams of venom to demonstrate its activity [40, 65, 124].
biological properties ascribed to the whole venom, The transcriptome analysis of L. laeta and L. intermedia
including dermonecrosis, massive inflammatory venom glands showed that this class of toxin is minim-
response with neutrophil infiltration, complement acti- ally expressed representing only 0.13% of the total
vation, platelet aggregation, immunogenicity, edema, expressed sequences of L. laeta venom gland [70, 72]. A
increased vessel permeability, hemolysis, renal failure, brown spider recombinant hyaluronidase from L. inter-
toxicity for several cultured cell types, and animal lethal- media venom presenting a molecular mass of 46 kDa
ity[65,76–81,84,90,92–95,114,117–120]. was obtained and characterized [122]. The active
Recently, we have observed that all this deleterious enzyme, after in vitro refolding, was able to degrade HA
events can be prevented using specific phospholipases and CS. These results corroborate previous data
inhibitors that can decrease the brown spider recombin- concerning a native hyaluronidase that degrades both
ant phospholipase-D activity [121]. This strengthen the glycosaminoglycans demonstrating that the recombinant
idea of the importance of designing and optimizing a hyaluronidase can also be considered as chondroitinase
specific drug to treat the serious clinical symptoms [122].Thebiologicalcharacterizationoftherecombinant
caused bythebrownspiderbite,apublic health problem hyaluronidase showed an increase in erythema, ecchym-
in several parts of the world and until now without osis, and dermonecrotic effects induced by the recom-
specific treatment. binant dermonecrotic toxin (LiRecDT1) in rabbit skin
[122]. Furthermore, a new Loxosceles intermedia
Productionofnovelandlessexpressedcomponentsin hyaluronidase isoform (42 kDa) was successfully
recombinantform expressed and secreted by insect cells (SF-9) by baculo-
Serineproteases, hyaluronidases, venom allergens, a virus technology. This novel toxin presented activity
histamine releasing factor also known as translationally against HA and its characterization is in process
controlled tumor protein (TCTP), enzymatic inhibitors (Chaves-Moreira:personal communication).
(serpins), and C-type lectins were identified in transcrip- The L. intermedia venom gland transcriptome analysis
tome studies of Loxosceles venom glands [70, 72]. The describedthe sequence ofaproteinidentified aspossible
cDNA libraries enabled an overview of the Loxosceles histamine releasing factor (HRF/TCTP) expressed at
venom and allowed the description of new molecules of relatively low level in the venom, i.e., only 0.4% of the
biotechnologicalinterest. toxin-encoding transcripts [72]. The functional
Since then, several components, i.e., TCTP and hyal- characterization of the recombinant protein, called
uronidases were further explored and produced as LiTCTP, revealed that this toxin leads to edema and
Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page8of12
enhanced vascular permeability [123]. The cutaneous applied. Eukaryotic expression systems are proposed to
symptoms of envenomation with Loxosceles venom in- ensure extraction of these toxins. Promising initial results
clude erythema, itching and pain. In some cases, Loxos- wereachievedwithbaculovirusandinsectcellstechnology
celes spider bites can cause hypersensitivity or even as well as with plant heterologous models for protein
allergic reactions. These responses could be associated expression, as these models promoted extraction of
with histaminergic events, such as an increase in vascu- soluble,pureandactiveformsofnewtoxins.
lar permeability and vasodilatation. LiTCTP could be Therefore, further studies focusing on the recombin-
associatedwiththesedeleterious venom activities,asthis ant production of novel toxins or the production of
protein was identified in L. intermedia venom. Another larger amounts of known toxins are imperative for
LoxoscelesTCTP has been described in the venom gland characterization of their different components. Loxos-
ofLoxosceleslaetausingtranscriptomeanalysis [70]. celes toxicology can explore the putative biotechno-
Sequences with significant similarity with allergen-like logical applications of toxins. The designing of inhibitor
toxins from other venoms were found on the transcrip- molecules for different toxins could be used as tools to
tome studies of L. laeta and L. intermedia venom glands elucidate the mechanisms of action and to elaborate
[70, 72]. These sequences described in L. intermedia protocols of basic and clinical research. It is of great
transcriptome encode for venom allergens that are interest to find inhibitors with the ability to stop or
cysteine-rich molecules and show significant similarity even delay the process of development and progression
to allergens from another spider genus (Lycosa sigorien- of loxoscelism as there is still no specific treatment
sis), scorpions and mite allergens [72]. The amino acid available for the brown spider bite.
sequence of a putative allergen from L. laeta venom is
Abbreviations
similar to venom allergen III and includes the presence
AGEP:Acutegeneralizedexanthematouspustulosis;C1P:Ceramide
of conserved cysteine residues [70]. In fact, allergic reac- 1-phosphate;CS:Condroitin-sulphate;HA:Hyaluronicacid;HRF:Histamine
tions following Loxosceles bites have been described in a releasingfactor;ICK:InhibitorCystineKnotfamily;IL-6:Interleukin-6;
KC:Keratinocytechemoattractant;LALP:Loxoscelesintermediaastacin-like
few cases, as reviewed by Gremski et al. in 2014 [10]. A
protease;LIBS1:Ligand-inducedbindingsite1;LPA:Releaseof
fine macular or papular eruption appears over the entire lysophosphatidicacid;LPC:Lysophosphatydilcholine;MCP-1:Monocyte
bodyinapproximately25%ofthepublishedcasesoflox- chemoattractantprotein-1;PLD:Phospholipase-D;SM:Sphingomyelin;
TCTP:Translationallycontrolledtumorprotein
oscelism. In addition, cases of acute generalized exan-
thematous pustulosis (AGEP) after accidents with L.
Acknowledgements
reclusa andL.rufescens havebeenreported [126, 127]. A ThanksareduetotheCenterfortheStudyofVenomsandVenomous
recombinant allergen factor from L. intermedia venom Animals(CEVAP)ofUNESPforenablingthepublicationofthispaper
(EditalToxinologiaCAPESno.063/2010,Processno.230.38.006285/2011-21,
was already cloned with a calculated molecular mass of
AUXPEToxinologia1219/2011).
46 kDa and five disulfide bonds (Chaves-Moreira:
personal communication). The expression of this recom- Funding
ThisworkwassupportedbytheEditalToxinologiaCAPESno.063/2010,
binant protein will help to investigate the underlying
Processno.23038.006268/2011-94,AUXPEToxinologia1216/2011.
mechanisms involved in the allergic responses observed
in loxoscelism cases and might be used to biomedical Authors’contributions
DCMcontributedtotheconceptionanddesignofstudy,andtothe
purposesinthis field.
acquisition,analysisand/orinterpretationofdata.DCM,LHG,ACMWand
OMCdraftedthemanuscript.ASR,OMCandSSVcriticallyrevisedthe
Conclusion manuscriptforimportantintellectualcontent.Allauthorsreadandapproved
thefinalmanuscript.
Loxosceles toxins are continuously being studied by
researchers worldwide (Figs. 1 and 2). In recent years, a Competinginterests
great amount of new toxins were identified in Loxosceles Theauthorsdeclarethattheyhavenocompetinginterests.
venom through combinationofdatafrommolecular biol-
Ethicsapprovalandconsenttoparticipate
ogytechniques,proteomicstudies,andcharacterizationof
Notapplicable.
recombinant toxins. Indeed, the identification, the bio-
chemicalandbiologicalcharacterizationandthestructural Authordetails
1DepartmentofCellBiology,FederalUniversityofParaná(UFPR),Curitiba,PR,
studies of Loxosceles toxins improved the knowledge on Brazil.2DepartmentofStructuralandMolecularBiology,StateUniversityof
venom composition and the involvement of these toxins PontaGrossa(UEPG),PontaGrossa,PR,Brazil.
inloxoscelism.However, therearemanymolecules(espe-
Received:8August2016Accepted:24January2017
cially, those with low level of expression) that remain
unidentified, without biological characterization and/or
unknown mechanisms of action. Most of these References
1. VetterRS,HinkleNC,AmesLM.Distributionofthebrownreclusespider
unidentifiedmoleculespresenteddifficultiesandsolubility
(Araneae:Sicariidae)inGeorgiawithcomparisontopoisoncenterreportsof
problems when prokaryotic expression systems were envenomations.JMedEntomol.2009;46:15–20.
Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page9of12
2. daSilvaPH,daSilveiraRB,AppelMH,MangiliOC,GremskiW,VeigaSS. 27. FigueiredoLF,Dias-LopesC,AlvarengaLM,MendesTM,Machado-de-Avila
Brownspidersandloxoscelism.Toxicon.2004;44:693–709. RA,McCormackJ,MinozzoJC,KalapothakisE,Chavez-OlorteguiC.
3. LucasSM.Thehistoryofvenomousspideridentification,venomextraction Innovativeimmunizationprotocolsusingchimericrecombinantproteinfor
methodsandantivenomproduction:alongjourneyattheButantan theproductionofpolyspecificloxoscelicantivenominhorses.Toxicon.
Institute,SaoPaulo,Brazil.JVenomAnimToxinsInclTropDis.2015;21:21. 2014;86:59–67.
4. CordeiroFA,AmorimFG,AnjoletteFA,ArantesEC.Arachnidsofmedical 28. JiacominiI,SilvaSK,AubreyN,MuzardJ,Chavez-OlorteguiC,DeMouraJ,
importanceinBrazil:mainactivecompoundspresentinscorpionand BillialdP,AlvarengaLM.Immunodetectionofthe“brown”spider(Loxosceles
spidervenomsandticksaliva.JVenomAnimToxinsInclTropDis. intermedia)dermonecrotoxinwithanscFv-alkalinephosphatasefusion
2015;21:24. protein.ImmunolLett.2016;173:1–6.
5. PlatinickNI.Theworldspidercatalog.BullAmMusNatHist.2013;14(5). 29. MacchiavelloA.Cutaneousarachnoidismexperimentallyproducedwiththe
6. FutrellJM.Loxoscelism.AmJMedSci.1992;304:261–7. glandularpoisonofLoxosceleslaeta.PRJPublicHealthTropMed.
7. HoganCJ,BarbaroKC,WinkelK.Loxoscelism:oldobstacles,newdirections. 1947;23:266–79.Spanishtransl280–293.
AnnEmergMed.2004;44:608–24. 30. VellardJ.ActioninvitroofthevenomoftheSouthAmericanspider
8. ChaimOM,Trevisan-SilvaD,Chaves-MoreiraD,WilleAC,FerrerVP, Loxosceleslaeta.CRHebdSeancesAcadSci.1956;243:825–6.
MatsubaraFH,MangiliOC,daSilveiraRB,GremskiLH,GremskiW,etal. 31. VellardJ.VenomofthespiderLoxosceleslaeta(Nic).CRHebdSeancesAcad
Brownspider(Loxoscelesgenus)venomtoxins:toolsforbiologicalpurposes. Sci.1956;243:433–6.
Toxins(Basel).2011;3:309–44. 32. DennyWF,DillahaCJ,MorganPN.HemotoxiceffectofLoxoscelesreclusus
9. Chaves-MoreiraD,Trevisan-SilvaD,GremskiLH,VeigaSS.Brownspider venom:invivoandinvitrostudies.JLabClinMed.1964;64:291–8.
venom:theidentificationandbiotechnologicalpotentialofvenomtoxins. 33. SmithCW,MicksDW.Acomparativestudyofthevenomandother
In:Venomgenomicsandproteomics.EditedbyGopalakrishnakoneP. componentsofthreespeciesofLoxosceles.AmJTropMedHyg.
Dordrecht:SpringerNetherlands;2014.p.1–22. 1968;17:651–6.
10. GremskiLH,Trevisan-SilvaD,FerrerVP,MatsubaraFH,MeissnerGO,Wille 34. MorganPN.Preliminarystudiesonvenomfromthebrownreclusespider
AC,VuitikaL,Dias-LopesC,UllahA,deMoraesFR,etal.Recentadvancesin Loxoscelesreclusa.Toxicon.1969;6:161–5.
theunderstandingofbrownspidervenoms:Fromthebiologyofspidersto 35. NormentBR,JarrattJH.Electrophoresisandelectrofocusingofvenomof
themolecularmechanismsoftoxins.Toxicon.2014;83:91–120. LoxoscelesreclusaGertsch&Mulaik(Araneida:Scytodidae).JMedEntomol.
11. IsbisterGK,FanHW.Spiderbite.Lancet.2011;378:2039–47. 1976;13:143–7.
12. ChippauxJP.Epidemiologyofenvenomationsbyterrestrialvenomous 36. GerenCR,ChanTK,HowellDE,OdellGV.Isolationandcharacterizationof
animalsinBrazilbasedoncasereporting:fromobviousfactsto toxinsfrombrownreclusespidervenom(Loxoscelesreclusa).ArchBiochem
contingencies.JVenomAnimToxinsInclTropDis.2015;21:13. Biophys.1976;174:90–9.
13. SINAN,SUS.CasosdeacidentesporaranhasnoBrasil.InMinistériodaSaúde 37. SuarezG,BiggemannU,SchenoneH.Biochemicalstudyofthevenomof
(NotificaçãoSdIdAded.,2015edition.Brasilia;2015. Loxosceleslaetaandofitsmechanismofaction.BolChilParasitol.
14. IsbisterGK,VetterRS.Loxoscelismandnecroticarachnidism:moremyths 1971;26:60–2.
andminorcorrections.AnnEmergMed.2005;46:205–6.authorreply 38. SuarezG,SchenoneH,SociasT.Loxosceleslaetavenom–partial
206–207. purification.Toxicon.1971;9:291.
15. DandoyC,GrimleyM.Secondaryhemophagocyticlymphohistiocytosis 39. WrightRP,ElgertKD,CampbellBJ,BarrettJT.Hyaluronidaseandesterase
(HLH)fromapresumedbrownreclusespiderbite.JClinImmunol. activitiesofthevenomofthepoisonousbrownreclusespider.Arch
2014;34:544–7. BiochemBiophys.1973;159:415–26.
16. SwansonDL,VetterRS.Loxoscelism.ClinDermatol.2006;24:213–21. 40. BordonKC,WiezelGA,AmorimFG,ArantesEC.Arthropodvenom
17. VetterRS,IsbisterGK.Medicalaspectsofspiderbites.AnnuRevEntomol. hyaluronidases:biochemicalpropertiesandpotentialapplicationsinmedicine
2008;53:409–29. andbiotechnology.JVenomAnimToxinsInclTropDis.2015;21:43.
18. MeneghelloJ,EmparanzaE.Cutaneo-visceralcomplicationsofLoxosceles 41. HeitzJR,NormentBR.Characteristicsofanalkalinephosphataseactivityin
laetabiteandcortisone;reportofacase.RevChilPediatr.1952;23:168–71. brownreclusevenom.Toxicon.1974;12:181–7.
19. DonckasterR,CohenH.AcaseofLoxoscelespoisoningdifficulttodiagnosis. 42. NormentBR,JongYS,HeitzJR.Separationandcharacterizationofvenom
BolChilParasitol.1960;15:81–2. componentsinLoxoscelesreclusa–III.Hydrolyticenzymeactivity.Toxicon.
20. SchenoneH,SemprevivoL,SchirmerE.Twocasesofcutaneo-visceral 1979;17:539–48.
loxoscelism.BolChilParasitol.1959;14:17–9. 43. JongYS,NormentBR,HeitzJR.Separationandcharacterizationofvenom
21. MalaqueCMS,ChaimOM,EntresM,BarbaroKC.Loxoscelesandloxoscelism: componentsinthebrownreclusespider(Loxoscelesreclusa)–I.Preparative
biology,venom,envenomation,andtreatment.In:GopalakrishnakoneP, discelectrophoresis.Toxicon.1979;17:307–12.
CorzoG,deLimaMH,Diego-GarcíaE,editors.Spidervenoms.Dordrecht: 44. JongYS,NormentBR,HeitzJR.Separationandcharacterizationofvenom
SpringerNature;2016.p.419–44. componentsinLoxoscelesreclusa–II.Proteaseenzymeactivity.Toxicon.
22. PauliI,MinozzoJC,daSilvaPH,ChaimOM,VeigaSS.Analysisoftherapeutic 1979;17:529–37.
benefitsofantiveninatdifferenttimeintervalsafterexperimental 45. BabcockJL,CivelloDJ,GerenCR.Purificationandcharacterizationofatoxin
envenomationinrabbitsbyvenomofthebrownspider(Loxosceles frombrownreclusespider(Loxoscelesreclusa)venomglandextracts.
intermedia).Toxicon.2009;53:660–71. Toxicon.1981;19:677–89.
23. MalaqueCM,SantoroML,CardosoJL,CondeMR,NovaesCT,RiskJY,Franca 46. ChuJY,RushCT,O'ConnorDM.Hemolyticanemiafollowingbrownspider
FO,deMedeirosCR,FanHW.Clinicalpictureandlaboratorialevaluationin (Loxoscelesreclusa)bite.ClinToxicol.1978;12:531–4.
humanloxoscelism.Toxicon.2011;58:664–71. 47. MoranO,ZavaletaA,CastrodelaMataR.HematologicaleffectsofLoxosceles
24. Dias-LopesC,GuimaraesG,FelicoriL,FernandesP,EmeryL,KalapothakisE, laetavenominalbinomice.BolChilParasitol.1981;36:20–3.
NguyenC,MolinaF,GranierC,Chavez-OlorteguiC.Aprotectiveimmune 48. BascurL,YevenesI,BarjaP.EffectsofLoxosceleslaetaspidervenomon
responseagainstlethal,dermonecroticandhemorrhagiceffectsof bloodcoagulation.Toxicon.1982;20:795–6.
Loxoscelesintermediavenomelicitedbya27-residuepeptide.Toxicon. 49. FoilLD,FrazierJL,NormentBR.Partialcharacterizationoflethaland
2010;55:481–7. neuroactivecomponentsofthebrownreclusespider(Loxoscelesreclusa)
25. Dias-LopesC,FelicoriL,RubrechtL,CoboS,MolinaL,NguyenC,GaleaP, venom.Toxicon.1979;17:347–54.
GranierC,MolinaF,Chavez-OlorteguiC.Generationandmolecular 50. Dias-LopesC,FelicoriL,GuimaraesG,GomesER,Roman-CamposD,Duarte
characterizationofamonoclonalantibodyreactivewithconservedepitope H,DamascenoD,MartinsM,KalapothakisE,AlmeidaAP,etal.Cardiotoxic
insphingomyelinasesDfromLoxoscelesspidervenoms.Vaccine. effectsofLoxoscelesintermediaspidervenomandtherecombinantvenom
2014;32:2086–92. toxinrLiD1.Toxicon.2010;56:1426–35.
26. RamadaJS,Becker-FincoA,MinozzoJC,FelicoriLF,MachadodeAvilaRA, 51. LucatoJrRV,AbdulkaderRC,BarbaroKC,MendesGE,CastroI,BaptistaMA,
MolinaF,NguyenC,deMouraJ,Chavez-OlorteguiC,AlvarengaLM. CuryPM,MalheirosDM,SchorN,YuL,BurdmannEA.Loxoscelesgaucho
Syntheticpeptidesforinvitroevaluationoftheneutralizingpotencyof venom-inducedacutekidneyinjury–invivoandinvitrostudies.PLoSNegl
Loxoscelesantivenoms.Toxicon.2013;73:47–55. TropDis.2011;5:e1182.
Chaves-Moreiraetal.JournalofVenomousAnimalsandToxinsincludingTropicalDiseases (2017) 23:6 Page10of12
52. NowatzkiJ,deSeneRV,PaludoKS,VeigaSS,OliverC,JamurMC,NaderHB, identificationofanewgroupofphospholipasesDfromLoxoscelessimilis
TrindadeES,FrancoCR.Brownspidervenomtoxinsinteractwithcell venomgland.Toxicon.2016;120:97–106.
surfaceandareendocytosedbyrabbitendothelialcells.Toxicon. 72. GremskiLH,daSilveiraRB,ChaimOM,ProbstCM,FerrerVP,NowatzkiJ,
2010;56:535–43. WeinschutzHC,MadeiraHM,GremskiW,NaderHB,etal.Anovel
53. NowatzkiJ,SeneRV,PaludoKS,RizzoLE,Souza-Fonseca-GuimaraesF,Veiga expressionprofileoftheLoxoscelesintermediaspidervenomousgland
SS,NaderHB,FrancoCR,TrindadeES.Brownspider(Loxoscelesintermedia) revealedbytranscriptomeanalysis.MolBiosyst.2010;2403–2416.
venomtriggersendothelialcellsdeathbyanoikis.Toxicon.2012;60:396–405. 73. WilleAC,Chaves-MoreiraD,Trevisan-SilvaD,MagnoniMG,Boia-FerreiraM,
54. CorreaMA,OkamotoCK,Goncalves-de-AndradeRM,vandenBergCW, GremskiLH,GremskiW,ChaimOM,Senff-RibeiroA,VeigaSS.Modulationof
TambourgiDV.SphingomyelinaseDfromLoxosceleslaetavenominduces membranephospholipids,thecytosoliccalciuminfluxandcellproliferation
theexpressionofMMP7inhumankeratinocytes:contributionto followingtreatmentofB16-F10cellswithrecombinantphospholipase-D
dermonecrosis.PLoSOne.2016;11:e0153090. fromLoxoscelesintermedia(brownspider)venom.Toxicon.2013;67:17–30.
55. BarbaroKC,LiraMS,AraujoCA,Pareja-SantosA,TavoraBC,Prezotto-Neto 74. MachadoLF,LaugesenS,BotelhoED,RicartCA,FontesW,BarbaroKC,
JP,KimuraLF,LimaC,Lopes-FerreiraM,SantoroML.Inflammatory RoepstorffP,SousaMV.Proteomeanalysisofbrownspidervenom:
mediatorsgeneratedatthesiteofinoculationofLoxoscelesgauchospider identificationofloxnecroginisoformsinLoxoscelesgauchovenom.
venom.Toxicon.2010;56:972–9. Proteomics.2005;5:2167–76.
56. TavaresFL,PeichotoME,RangelDdeM,BarbaroKC,CirilloMC,SantoroML, 75. KalapothakisE,ChatzakiM,Goncalves-DornelasH,deCastroCS,SilvestreFG,
Sano-MartinsIS.Loxoscelesgauchospidervenomanditssphingomyelinase LaborneFV,deMouraJF,VeigaSS,Chavez-OlorteguiC,GranierC,Barbaro
fractiontriggerthemainfunctionsofhumanandrabbitplatelets.HumExp KC.TheLoxtoxproteinfamilyinLoxoscelesintermedia(Mello-Leitao)venom.
Toxicol.2011;30:1567–74. Toxicon.2007;50:938–46.
57. TavaresFL,PeichotoME,MarcelinoJR,BarbaroKC,CirilloMC,SantoroML, 76. AppelMH,daSilveiraRB,ChaimOM,PaludoKS,SilvaDT,ChavesDM,da
Sano-MartinsIS.Plateletparticipationinthepathogenesisofdermonecrosis SilvaPH,MangiliOC,Senff-RibeiroA,GremskiW,etal.Identification,cloning
inducedbyLoxoscelesgauchovenom.HumExpToxicol.2016;35:666–76. andfunctionalcharacterizationofanoveldermonecrotictoxin
58. BarbaroKC,CardosoJL,EickstedtVR,MotaI.Dermonecroticandlethal (phospholipaseD)frombrownspider(Loxoscelesintermedia)venom.
componentsofLoxoscelesgauchospidervenom.Toxicon.1992;30:331–8. BiochimBiophysActa.2008;1780:167–178.
59. TambourgiDV,MagnoliFC,vandenBergCW,MorganBP,deAraujoPS,Alves 77. ChaimOM,SadeYB,daSilveiraRB,TomaL,KalapothakisE,Chavez-
EW,DaSilvaWD.SphingomyelinasesinthevenomofthespiderLoxosceles OlorteguiC,MangiliOC,GremskiW,vonDietrichCP,NaderHB,Sanches
intermediaareresponsibleforbothdermonecrosisandcomplement- VeigaS.Brownspiderdermonecrotictoxindirectlyinducesnephrotoxicity.
dependenthemolysis.BiochemBiophysResCommun.1998;251:366–73. ToxicolApplPharmacol.2006;211:64–77.
60. FeitosaL,GremskiW,VeigaSS,EliasMC,GranerE,MangiliOC,BrentaniRR. 78. daSilveiraRB,PigozzoRB,ChaimOM,AppelMH,DreyfussJL,TomaL,
Detectionandcharacterizationofmetalloproteinaseswithgelatinolytic, MangiliOC,GremskiW,DietrichCP,NaderHB,VeigaSS.Molecularcloning
fibronectinolyticandfibrinogenolyticactivitiesinbrownspider(Loxosceles andfunctionalcharacterizationoftwoisoformsofdermonecrotictoxin
intermedia)venom.Toxicon.1998;36:1039–51. fromLoxoscelesintermedia(brownspider)venomgland.Biochimie.
61. VeigaSS,daSilveiraRB,DreyfusJL,HaoachJ,PereiraAM,MangiliOC, 2006;88:1241–53.
GremskiW.Identificationofhighmolecularweightserine-proteasesin 79. daSilveiraRB,PigozzoRB,ChaimOM,AppelMH,SilvaDT,DreyfussJL,
Loxoscelesintermedia(brownspider)venom.Toxicon.2000;38:825–39. TomaL,DietrichCP,NaderHB,VeigaSS,GremskiW.Twonovel
62. YoungAR,PincusSJ.Comparisonofenzymaticactivityfromthreespecies dermonecrotictoxinsLiRecDT4andLiRecDT5frombrownspider(Loxosceles
ofnecrotisingarachnidsinAustralia:Loxoscelesrufescens,Badumnainsignis intermedia)venom:fromcloningtofunctionalcharacterization.Biochimie.
andLamponacylindrata.Toxicon.2001;39:391–400. 2007;89:289–300.
63. daSilveiraRB,dosSantosFilhoJF,MangiliOC,VeigaSS,GremskiW,Nader 80. VuitikaL,GremskiLH,Belisario-FerrariMR,Chaves-MoreiraD,FerrerVP,Senff-
HB,vonDietrichCP.Identificationofproteasesintheextractofvenom RibeiroA,ChaimOM,VeigaSS.Brownspiderphospholipase-Dcontaininga
glandsfrombrownspiders.Toxicon.2002;40:815–22. conservativemutation(D233E)inthecatalyticsite:identificationandfunctional
64. ZanettiVC,daSilveiraRB,DreyfussJL,HaoachJ,MangiliOC,VeigaSS, characterization.JCellBiochem.2013;114:2479–92.
GremskiW.Morphologicalandbiochemicalevidenceofbloodvessel 81. LeeS,LynchKR.Brownreclusespider(Loxoscelesreclusa)venom
damageandfibrinogenolysistriggeredbybrownspidervenom.Blood phospholipaseD(PLD)generateslysophosphatidicacid(LPA).BiochemJ.
CoagulFibrinolysis.2002;13:135–48. 2005;391:317–23.
65. BarbaroKC,KnysakI,MartinsR,HoganC,WinkelK.Enzymatic 82. Ramos-CerrilloB,OlveraA,OdellGV,ZamudioF,Paniagua-SolisJ,AlagonA,
characterization,antigeniccross-reactivityandneutralizationof StockRP.GeneticandenzymaticcharacterizationofsphingomyelinaseD
dermonecroticactivityoffiveLoxoscelesspidervenomsofmedical isoformsfromtheNorthAmericanfiddlebackspidersLoxoscelesbonetiand
importanceintheAmericas.Toxicon.2005;45:489–99. Loxoscelesreclusa.Toxicon.2004;44:507–14.
66. deCastroCS,SilvestreFG,AraujoSC,deMYG,MangiliOC,CruzI,Chavez- 83. LajoieDM,RobertsSA,Zobel-ThroppPA,DelahayeJL,BandarianV,Binford
OlorteguiC,KalapothakisE.Identificationandmolecularcloningof GJ,CordesMH.VariableSubstratepreferenceamongphospholipaseD
insecticidaltoxinsfromthevenomofthebrownspiderLoxosceles toxinsfromSicariidspiders.JBiolChem.2015;290:10994–1007.
intermedia.Toxicon.2004;44:273–80. 84. CatalanA,CortesW,SaguaH,GonzalezJ,ArayaJE.Twonewphospholipase
67. Fernandes-PedrosaF,JunqueiradeAzevedoIdeL,Goncalves-de-Andrade DisoformsofLoxosceleslaeta:cloning,heterologousexpression,functional
RM,vandenBergCW,RamosCR,HoPL,TambourgiDV.Molecularcloning characterization,andpotentialbiotechnologicalapplication.JBiochemMol
andexpressionofafunctionaldermonecroticandhaemolyticfactorfrom Toxicol.2011;25:393–403.
Loxosceleslaetavenom.BiochemBiophysResCommun.2002;298:638–45. 85. deSantiFerraraGI,Fernandes-PedrosaMdeF,Junqueira-de-AzevedoIdeL,
68. KalapothakisE,AraujoSC,deCastroCS,MendesTM,GomezMV,Mangili Goncalves-de-AndradeRM,PortaroFC,Manzoni-de-AlmeidaD,Murakami
OC,GubertIC,Chavez-OlorteguiC.Molecularcloning,expressionand MT,ArniRK,vandenBergCW,HoPL,TambourgiDV.SMaseII,anew
immunologicalpropertiesofLiD1,aproteinfromthedermonecroticfamily sphingomyelinaseDfromLoxosceleslaetavenomgland:molecularcloning,
ofLoxoscelesintermediaspidervenom.Toxicon.2002;40:1691–9. expression,functionandstructuralanalysis.Toxicon.2009;53:743–53.
69. MagalhaesGS,CaporrinoMC,Della-CasaMS,KimuraLF,Prezotto-NetoJP, 86. DesaiA,LankfordHA,WarrenJS.Loxoscelesdesertaspidervenominduces
FukudaDA,Portes-JuniorJA,Neves-FerreiraAG,SantoroML,BarbaroKC. theexpressionofvascularendothelialgrowthfactor(VEGF)inkeratinocytes.
Cloning,expressionandcharacterizationofaphospholipaseDfrom Inflammation.2000;24:1–9.
Loxoscelesgauchovenomgland.Biochimie.2013;95:1773–83. 87. DesaiA,MillerMJ,GomezHF,WarrenJS.Loxoscelesdesertaspidervenom
70. Fernandes-PedrosaF,Junqueira-de-AzevedoL,Goncalves-de-AndradeRM, inducesNF-kappaB-dependentchemokineproductionbyendothelialcells.J
KobashiLS,AlmeidaDD,HoPL,TambourgiDV.Transcriptomeanalysisof ToxicolClinToxicol.1999;37:447–56.
Loxosceleslaeta(Araneae,Sicariidae)spidervenomousglandusing 88. ChatzakiM,HortaCC,AlmeidaMO,PereiraNB,MendesTM,Dias-LopesC,
expressedsequencetags.BMCGenomics.2008;9:279. GuimaraesG,MoroL,Chavez-OlorteguiC,HortaMC,KalapothakisE.
71. DantasAE,CarmoAO,HortaCC,LealHG,Oliveira-MendesBB,MartinsAP, CutaneousloxoscelismcausedbyLoxoscelessimilisvenomand
Chavez-OlorteguiC,KalapothakisE.DescriptionofLoxtoxproteinfamilyand neutralizationcapacityofitsspecificantivenom.Toxicon.2012;60:21–30.