antigenic, microbicidal and antiparasitic properties of an l-amino

8/12/2019 Antigenic, Microbicidal and Antiparasitic Properties of an L-Amino

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Antigenic, microbicidal and antiparasitic properties of an L-aminoacid oxidase isolated from Bothrops jararaca snake venom

P. Ciscotto a, R.A. Machado de Avila a, E.A.F. Coelho a, J. Oliveira a, C.G. Diniz b, L.M. Faras b,M.A.R. de Carvalho b, W.S. Maria c, E.F. Sanchez c, A. Borges d, C. Chavez-Olortegui a,*

a Departamento de Bioqumica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627,

30161-970 Belo Horizonte, Minas Gerais, Brazilb Departamento de Microbiologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazilc Fundaao Ezequiel Dias, Rua Conde Pereira Carneiro 80, 30550-010 Belo Horizonte, MG, Brazild Laboratorio de Toxinas Animales, Centro de Biociencias y Medicina Molecular, Instituto de Estudios Avanzados, Apartado 17606, Caracas 1015-A, Venezuela

a r t i c l e i n f o

Article history:

Received 2 September 2008

Received in revised form 3 December 2008

Accepted 4 December 2008

Available online 11 December 2008

Keywords:

Bothrops jararaca

L-Amino acid oxidase

MicrobicidalLeishmanicidal

Neutralizing potency

a b s t r a c t

Venoms from the beeApis mellifera, the caterpillar Lonomia achelous, the spidersLycosasp.

and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the

snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni,

Bothrops neuwiedi,Crotalus durissus terrificus, andLachesis mutawere assayed (800mg/mL)

for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu

showed the highest S. aureus growth inhibition and also against other Gram-positive and

Gram-negative bacteria. To characterize the microbicidal component(s) produced by

B. jararaca, venom was fractionated through gel exclusion chromatography. The high

molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchangechromatography through Mono Q columns using a 00.5 M NaCl gradient. Bactericidal

Mono Q fractions P5 and P6 showed significant LAAO activity using L-leucine as substrate.

These fractions were pooled and subjected to Heparin affinity chromatography, which

rendered a single LAAO activity peak. The anti-S. aureusactivity was abolished by catalase,

suggesting that the effect is dependent on H2O2 production. SDS-PAGE of isolated LAAO

indicated the presence of three isoforms since deglycosylation with a recombinant

N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was

142.7 U/mg, based on the oxidation of L-leucine. The correlation between in vivo

neutralization of lethal toxicity (ED50) and levels of horse therapeutic antibodies anti-LAAO

measured by ELISA was investigated to predict the potency of Brazilian antibothropic

antivenoms. Six horses were hyperimmunized with Bothropsvenoms (50% fromB. jararaca

and 12.5% each from B. alternatus, B. jararacussu,B. neuwiediiand B. moojeni). To set up an

indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation

coefficients (r) between ED50and ELISA antibody titers againstB. jararaca venom and LAAO

were 0.846 (p


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ammonia. LAAOs are amongst the most abundant proteinsin ophidian, particularly hemorrhagic venoms (Du andClemetson, 2002; Guercio et al., 2006) and are capable ofinducing apoptosis of various cell types, including vascularendothelial cells (VEC). Although the apoptosis mechanismis not yet clear, it involves the production of H2O2which isachieved by oxidation of some VEC plasma membraneproteins (Suhr and Kim,1996; Torii et al.,1997, 2000). Otheractivities from LAAOs include induction or inhibition ofplatelet aggregation (Li et al., 1994; Sakurai et al., 2001; Luet al., 2002), anticoagulant activity (Sakurai et al., 2003),stimulation of edema formation (Wei et al., 2002; Stabeliet al., 2004; Izidoro et al., 2006), hemorrhage (Stabeli et al.,2004) and antibacterial, antiviral, and leishmanicidalfunctions (Lu et al., 2002; Wei et al., 2002; Zhang et al.,2003; Izidoro et al., 2006). Venoms from viperid snakesbelonging to the Neotropical genus Bothrops are speciallyenriched in LAAOs (Pessatti et al., 1995; Tan and Ponndurai,1991). Significant antitumoral (da Silva et al., 2002a,b),anti-Leishmania major, and anti- Trypanosoma cruzi(Gonalves et al., 2002) activities have been found invenom from the BrazilianBothrops jararaca, which could berelated to its LAAO-mediated production of H2O2, as hasbeen shown for Bothrops moojeni (Tempone et al., 2001).LAAOs from Bothrops alternatus (Stabeli et al., 2004), B.moojeni(Stabeli et al., 2007), and Bothrops pirajai (Izidoroet al., 2006) have been characterized biochemically andfunctionally.

Given the diversity of LAAOs in terms of molecular mass,substrate specificity, interaction with platelets, induction ofhemorrhage and apoptosis, and antibacterial and antipar-asitic activities (Du and Clemetson, 2002), we undertookthe characterization of the enzyme from B. jararaca not onlyfor a better understanding of its role in the envenomingmechanism, but also its biotechnological potential asimmunochemical reagent to develop anin vitrotechniquefor estimating the neutralizing potency of horse Brazilianantibothropic antivenoms. Antivenoms are considered tobe the only specific treatment for envenoming by snakes.These therapeutic antivenoms are traditionally preparedfrom hyperimmunized horse plasmas. The neutralizationability of snake antivenoms is still assessed by the tradi-tional in vivo lethality assay (minimum effective dose:ED50), performed in mice (World Health Organization(WHO), 1981). Besides its inherent aggressiveness to theanimals, this procedure is expensive, cumbersome, andtime consuming. Reproducibility is quite difficult toachieve, and it is strongly dependent on qualified andtrained personnel. In vitro alternative assays that mayreplace, at least in part, the use of animals are urgentlysought and is the main subject of this paper. Enzyme-linkedimmunosorbent assays (ELISAs) have been used in studiesto assess antivenom potency against snakes (Theakston andReid, 1979; Rungsiwongse and Ratanabanangkoon, 1991;Barbosa et al., 1995; Heneine et al., 1998; Rial et al., 2006)and scorpion venoms (Maria et al., 2005).

In this work, a screening of the microbicidal potency ofthirteen insect, arachnid and ophidian venoms renderedB. jararaca as the most active. Such potency was relatedto its LAAO activity, which was isolated and furthercharacterized biochemically. B. jararaca LAAO is a potent

microbicidal (anti-Staphylococcus aureus) and leishmanici-dal (anti-Leishmania amazonensis) component with glyco-sylated isoforms which differ from the snake venom LAAOsstudied thus far. We have shown also, that LAAO can beused as antigen coated to ELISA plates to develop anin vitrotechnique for estimating the potency of horse Brazilianantibothropic antivenoms.

2. Materials and methods

2.1. Venoms and antivenoms

Crude venoms from the beeApis mellifera, the caterpillarLonomia achelous, the spiders Lycosa sp. and Phoneutrianigriventer, the scorpions Tityus bahiensis and Tityusserrulatus, the snakes B. alternatus, B. jararaca, Bothrops

jararacussu,B. moojeni,Bothrops neuwiedi,Crotalus durissusterrificus and Lachesis muta muta were provided by theSeao de Animais Peonhentos of Fundaao Ezequiel Dias(FUNED), Belo Horizonte, Minas Gerais, Brazil and keptlyophilized at 20 C, at the Laboratory of Toxin Immuno-chemistry, Instituto de Ciencias Biologicas, UniversidadeFederal de Minas Gerais, Belo Horizonte, Brazil. Hyperim-mune horse antibothropic plasmas (n 6) were obtainedfollowing the standard immunization schedules of theImmunobiological Production Unit at FUNED. The antigenfor producing the antibothropic plasmas consisted of 50% ofB. jararacavenom and 12.5% each ofB. alternatus, B. jarar-acussu,B. neuwiediiandB. moojenivenoms. One week afterthe last injection of antigen, all horses were bled by veni-puncture. Blood samples were kept in test tubes and, afterclotting, the serum was separated and stored at 20 C.

2.2. Protein determination

Protein content in crude venoms and isolated fractionswere determined according to the method of Bradford(1976) utilizing bovine serum albumin (Sigma Chemicals)as a standard.

2.3. Antibacterial activity

Agar diffusion assays for bactericidal activity werecarried out at the Laboratory of Oral Microbiology, Depart-ment of Microbiology, Universidade Federal de MinasGerais,according to the agar diffusion method described byNational Committee for Clinical Laboratory Standards(2001). Briefly, bacteria were suspended in saline (0.85% w/v) and homogenously inoculated on Petri dishes containingBrain-Heart Infusion media (5.2% w/v) and yeast extract(0.5% w/v). Depending on the nutritional requirements ofthe tested microorganism, the growth medium was sup-plemented with 5mg/mL of menadione and hemine. Proteinsamples (40mg in 50 mL of saline) were applied onto wellsmolded on the agar. Fifty mL of potassium metabisulphite(50 mg/mL) and NaCl 0.87% w/v were used as positive andnegative controls, respectively. Plates were incubated at37 C until bacterial growth was detected. Bactericidalactivity was assessed according to the inhibition haloformed around the well. All experiments were performed induplicates using separate plates. Various Gram-positive and

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Gram-negative bacteria were tested for their growth sensi-tivity upon incubation with various venoms. Aerobicbacteria were Escherichia coli (ATCC 25922), Listeria mono-cytogenes (ATCC 15313), Pseudomonas aeruginosa (ATCC10145), Salmonella typhimurium (ATCC 14028), S. aureus(ATCC 33591), Staphylococcus epidermidis (ATCC 12228),which were incubated at 37 C, whereas anaerobic orfacultative bacteria (Actinobacillus actinomycetencomitans(ATCC 29523) Bacteroides fragilis (ATCC 25285), Eikenellacorrodes, Enterococcus fecalis (ATCC 19433), Eubacteriumlentum, Peptostreptococcus anaerobius (ATCC 27337),Porphyromonas gingivalis (ATCC 33277), Prevotella inter-media (ATCC 25611), Propionibacterium acnes (ATCC 6919)andStreptococcus mutans(ATCC 25175)), were incubated inanaerobic chambers at 37 C.

2.4. Polyacrylamide gel electrophoresis in

the presence of SDS (SDS-PAGE)

Protein samples were subjected to SDS-PAGE according

to the method ofLaemmli (1970)in the absence or pres-ence (reducing conditions) ofb-mercaptoethanol.

2.5. L-Amino acid oxidase (LAAO) activity

LAAO activity was assessed in B. jararaca chromato-graphic fractions or crude venom according to the methodofSakurai et al. (2001)with modifications. Briefly, a reac-tion mixture containing horseradish peroxidase (50mg/mL),100mM L-leucine, 10mM o-dianisidine in 100 mM TrisHCl (pH 7.8), and the experimental sample (2mg) in a finalvolume of 1 mL was incubated at 25 C for 30 min. Theincrease in absorbance at 436 nm was recorded in

a Shimadzu UV 160A spectrophotometer. One unit ofenzymatic activity is defined as the oxidation of 1mML-leucine per min. Assays were performed in duplicates.The following substrates were used to determine thesubstrate specificity of the isolated B. jararaca LAAO:L-alanine, L-arginine, L-asparagine, L-cysteine, L-phenylala-nine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methio-nine, L-proline, L-threonine, L-tryptophan, L-serine, andL-valine. The reaction mixture was set as described above,at an amino acid concentration of 100mg/mL and 2.0mg/mLofB. jararacaLAAO.

2.6. Chromatographic separation ofB. jararacacrude venom

2.6.1. Gel filtration chromatography

Crude B. jararacavenom was initially resolved througha gel filtration semipreparative column (Superdex 75HR10/30 Pharmacia, 10 mm diameter) operated by a FastPerformance Liquid Chromatography (FPLC, Pharmacia)system. Venom (500 mg protein) was solubilized in 0.5 mLof elution buffer (0.15 M ammonium formiate, pH 6.0) andapplied to the equilibrated column (10 mg per chromato-graphic run), which was subsequently eluted at 0.5 mL/min, and the eluate collected in 1-mL fractions. Absorbancewas recorded at 280 nm. Fractions were pooled accordingto the chromatographic profile, lyophilized and stored at80 C until performance of the bactericidal assays.

2.6.2. Anion exchange chromatography

The gel filtration chromatography fraction exhibitingbactericidal activity was solubilized in elution buffer(20 mM HEPES, pH 8.0), and applied onto an anionexchange column (Mono QR HR 5/5, Pharmacia), operatedby an FPLC (Pharmacia) system (5 mg protein per run). Atotal of 147 mg of protein was fractionated. The Mono Qcolumn was equilibrated with elution buffer and eluted at1 mL/min using a linear 00.5 M NaCl gradient. Absorbanceof the eluate (collected in 1 mL fractions) was recorded at280 nm. Fractions were pooled according to the chro-matographic profile, lyophilized and stored at 80 C untilperformance of the bactericidal assays.

2.6.3. Affinity chromatography through HiTrap

Heparin columns

Anion exchange chromatography fractions exhibitingbactericidal activity were pooled and dialyzed against5 mM Mes, 5 mM Tris, 1 mM Benzamidine, pH 6.0 (solutionA), at 4 C for 18 h. Subsequently, the protein sample(11 mg) was applied onto a HiTrap Heparin column(0.7 2.5 cm) previously equilibrated with solution A. Thecolumn was then developed with a NaCl linear (00.2 M)gradient. Fractions (1 mL) were collected at a flow rate of0.5 mL/min at room temperature. Absorbance was recor-ded at 280 nm. All fractions were assayed for LAAO activityaccording to the procedure outlined in Section 2.5. Theactive fractions were pooled, lyophilized and stored at80 C until performance of bactericidal activity assays.

2.7. Deglycosylation ofB. jararacaLAAO

LAAO obtained from affinity chromatography columns asdescribed in Section 2.6 was subjected to deglycosylation bytreating the enzyme with recombinant Glycosidase F fromFlavobacterium meningosepticum(PNGase F) as previouslydescribed by Magalhaes et al. (2007). Briefly, 200mg ofpurified protein were dissolved in 90mL of denaturing buffer(0.5% SDS, 1%m-mercaptoethanol) and denatured by boilingfor 10 min. Tenml of reaction buffer (0.05 M phosphate, pH7.5) was then added, together with 10mL of 10% NP-40 and2ml of recombinant PNGase F. The resulting digestion wasanalyzed by SDS-PAGE as described in Section2.4.

2.8. Leishmanicidal activity

Strain IFLA/BR/1967/pH-8 of Leishmania (Leishmania)amazonensis was used. The assay for antiparasitic activitywas carried out according to the procedure ofTempone et al.(2001) with major modifications. Briefly, promastigoteswere cultured in Schneiders complete medium (SigmaChemicals), supplemented with 20% inactivated fetal bovineserum (Sigma), 20 mM L-glutamine, 50mg/mL gentamicin,200 U/mL penicillin and 100mg/mL streptomycin, at pH 7.4.Parasites were cultured at 23 C for 5 days. Viability wasassessed by quantification in a Neubauer chamber. L. (L.)amazonensis promastigotes were incubated in 96-wellCostar microtitration plates at 4 105 cells/well, togetherwith the experimental sample at 0.8 mg/mL, in the absenceor presence of catalase (0.3 mg/mL). Incubation was per-formed in Dulbeccos Modified Eagles Medium (DMEM,

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Sigma) containing 20% inactivated fetal bovine serum, 4.5 g/L glucose, 20mg/mL gentamicin sulphate, 100 U/mL peni-cillin and 50mg/mL streptomycin at pH 7.4, in a final volumeof 150 mL/well for 18 h at 23 C. Negative (without sample)and positive (with H2O2) controls were also included.Promastigotes were also incubated in complete HanksBalanced Salt Solution (HBSS), characterized by its pooraminoacid content, under the same conditions. Cell viability wasassessed by the oxidation of MTT (3-[4,5-dimethylthiazol-2-1]-2,5diphenyl-tetrazolium bromide) according toMachadoet al. (2007). Results are the mean of two independentexperiments performed in triplicates.

2.9. Hemolytic activity

Hemolytic activity was determined in TSA solid culturemedium (1.5% bacto tryptone, 0.5% bacto soytone, 0.5%NaCl, 1.5% bacto agar) containing 5% horse blood. Samples(in 50mL) were applied onto wells made on the solidifiedmedium and the plates were incubated at 37 C for 6 h,

when the erythrocyte lysis could be detected by theformation of a halo. Hemolysis areas were characterized asb-hemolysis, partial hemolysis, or b-hemolysis zones.Hemolytic activity assays were performed in duplicates.

2.10. Lethality ofB. jararacavenom and in vivo

neutralization assays

LethalityofB. jararaca crude venomwas determined by i.p.injection into Swiss mice. The LD50 ofB. jararaca crude venom,used throughout this study was 42mg per 20 g mouse weight.Deaths were recorded up to 48 h. A fixed amount (5 LD50) ofBothropsvenom was incubated with varying amounts of therespective antivenom for 30 min at 37 C. Each mixture(0.5 mL) was injected i.p. into groups of eight Swiss mice(18 22 g) and deaths were recorded up to 48 h. Results wereanalyzed using the Probit test and neutralization wasexpressed as effective dose 50% (ED50), defined as the amount(mg) of venom neutralized by 1.0 mL of antivenom (volumeneeded to prevent death in 50% of the injected mice).

2.11. ELISA

ELISA was performed by coating the plates (Nunc/Denmark) overnight at 5 C with 100 ng/well of crudeB. jararaca venom. After blocking and washing, the horseantibothropic sera corresponding to a final dilution of1:40,000 were added and incubated for 1 h at 37 C. ELISAwas performed by the method ofMaria et al. (1998). Theabsorbance values were determined at 492 nm witha Titertek Multiscan spectrophotometer. All measurementswere made in duplicate and the results were expressed asthe median of two assays. Correlation and regressionanalysis were performed by the least-squares meansmethod using standard software (Excel and Instat).

3. Results

3.1. Screening of insect, arachnid, and ophidian

venoms for bactericidal activity

We assayed for antibacterial activity of thirteen venomsfrom various insect, arachnid and ophidian sourcesutilizingS. aureus as target bacterium.Fig. 1shows repre-sentative plates with inhibition haloes indicating thatsnake venoms from the genusBothropsare the most potentanti-S. aureus bactericidal agents at the concentrationtested. Noticeably, venoms from B. jararacussu andB. jararacacontained the highest activity.

3.2. Gram-positive and Gram-negative bacteria sensitivity

toB. jararacussu and B. jararacavenoms

Given that the anti-S. aureus tests rendered B. jarar-acussuand B. jararaca as the bothropic venoms exhibitingthe highest bactericidal activity, both venoms were assayedagainst an ample spectrum of representative Gram-positiveand Gram-negative bacterial strains. Table 1 shows theresults of inhibition tests performed on seven Gram-positive and seven Gram-negative bacteria responsible forvarious human pathologies. The venoms were capable of

Fig. 1. Growth inhibition of S. aureus by insect, arachnid, and snake venoms (0.8 mg/mL). 1. Apis mellifera, 2. Lonomia achelous, 3. Lycosa sp., 4. Phoneutria

nigriventer, 5. Tityus bahiensis, 6.T. serrulatus, 7.Lachesis muta muta, 8. Bothrops jararacussu, 9. B. alternatus, 10. B. moojeni, 11.B. neuwiedii, 12. Crotalus durrisus

terrificus,13.B. jararaca. (C

) Positive control, potassium metabisulphite (50 mg/mL). (C

) Negative control, NaCl 0.87% w/v. Plates were incubated for approx. 7 hat 37 C.



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equally inhibiting the growth of five Gram-positive andfour Gram-negative species. Noticeably, E. faecalis andE. coliwere resistant to both venoms at the dose tested.

3.3. Gel filtration chromatography ofB. jararacavenom

Based on the anti-S. aureusactivity ofB. jararacavenom(Fig. 1), we undertook its fractionation by gel filtrationchromatography to initiate isolation and characterizationof the bactericidal component(s).Fig. 2A shows the elutionprofile through Superdex columns (equilibrated in 0.15 Mammonium formiate, pH 6.0) ofB. jararacasoluble venom

(10 mg). A total of 500 mg of protein were fractionatedusing this protocol. The high molecular weight fraction P1contained approx. 30% ofB. jararacawhole venom bacte-ricidal (anti-S. aureus) activity (Fig. 2B). P1 exhibited LAAOactivity (approx. 80% of the whole venom activity), basedon the assay for oxidation ofL-leucine (Fig. 2C).

3.4. Anion exchange chromatography

To further purify the bactericidal components withLAAO activity, gel filtration fraction P1 (5 mg) was sus-pended in 20 mM HEPES buffer (pH 8.0) and applied ontoa Mono Q HR 5/5 column previously equilibrated with the

same solution. A total of 140 mg of fraction P1 werefractionated using such protocol.Fig. 3A shows the chro-matographic profile of P1 proteins eluted using a linear00.5 M NaCl gradient. LAAO activity was contained infractions P5 and P6 which together correspond to 8% of theprotein content of gel filtration fraction P1. P5 and P6 werepooled into fraction TAP5/6 for further fractionationprocedures. TAP5/6 retained most of the bactericidalactivity of fraction P1 (Fig. 3B).

3.5. Affinity chromatography

Active anion exchange fraction TAP5/6 (11 mg) was dia-lyzed against 10 mM Mes, 10 mM Tris, 2 mM Benzamidine

(pH 6.0) and applied onto Heparin columns for affinitychromatography on HiTrap Heparin columns, equilibratedwith the same solution and eluted with a linear 00.4 MNaCl gradient. This approach has been used previously forisolation of LAAOs from other snake venoms.Fig. 4A showsthe elution profile, indicating that LAAO activityeluted in thefraction 1 from the column. This fraction was named HTP1.After dialysis against 0.05 M TrisHCl (pH 7.8), it wasconfirmed that HTP1 was active againstS. aureus(Fig. 4B).With the goal of determining whether HTP1 bactericidalactivity was related to the hydrogen peroxide produced viaLAAO, anti-S. aureusactivity was determined after incuba-

tion of the fraction with catalase (0.3 mg/mL). Catalase (EC1.11.1.6) catalyzes the conversion of H2O2to water and O2. Itwas found that the HTP1 bactericidal activity was abolishedafter such incubation with catalase (Fig. 4B, well 3).

3.6. Electrophoretic composition of purified fractions

and effect of deglycosylation

To assess the purity and subunit composition of thebactericidal, LAAO-active HTP1 fraction obtained byaffinity chromatography, SDS-PAGE gels were performed.Fig. 5 shows the presence of three distinct bands withmolecular masses 80, 60.8 and 48.1 kDa (Fig. 5A). Todetermine whether the above components could beglycosylated by variants of a unique B. jararaca LAAOenzyme, we subjected fraction HTP1 to digestion withGlycosidase (PNGase) F (see Section 2.7) and electro-phoresed by SDS-PAGE. Such procedure has been usedpreviously to study the glycan moieties of LAAO fromCalloselasma rhodostoma (Geyer et al., 2001). PNGase F isan amidase that cleaves between the innermost GlcNAcand asparagine residues of high mannose, hybrid, andcomplex oligosaccharides from N-linked glycoproteins(Maley et al., 1989). After digestion of HTP1, a singleelectrophoretic component of 38.2 kDa was obtained(Fig. 5B), attesting to the purity of the fraction.

Table 1

Sensitivity of Gram-positive and Gram-negative bacteria to B. jararacaand B. jararacussuvenoms; () bacterial inhibition; () without bacterial inhibition.

Bacterial strain Inhibition of bacterial growth

byBothropsvenomsaPathology

B. jararaca B. jararacussu

Gram-positive Eubacterium lentum Urine infection

Peptostreptococcus anaerobius(ATCC 27337) Respiratory tract infection

Propionibacterium acnes(ATCC 6919) Skin infection

Staphylococcus aureus(ATCC 33591) Hospitalary infectionStaphylococcus epidermidis(ATCC 12228) Skin infection

Enterococcus fecalis(ATCC 19433) Hospitalary infection

Streptococcus mutans (ATCC 25175) Periodontal disease

Gram-negative Porphyromonas gingivalis(ATCC 33277) Periodontal disease

Prevotella intermedia(ATCC 25611) Periodontal disease

Pseudomonas aeruginosa(ATCC 10145) Hospitalary infection

Salmonella typhimurium(ATCC 14028) Food poisoning

Bacteroides fragilis(ATCC 25285) Skin infection

Eikenella corrodes Respiratory tract infection

Escherichia coli(ATCC 25922) Food poisoning

a Assays were performed at 0.8 mg/mL venom concentration.

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3.7. Leishmanicidal and hemolytic activity ofB. jararaca

crude venom and LAAO-active fractions

Taking into account the observed bactericidal activity offraction HTP1 (also identified as an LAAO-active fraction),we wished to determine its antiparasitic (leishmanicidal)and hemolytic potency. It is known that other ophidian

LAAOs exhibit such activities as a result of enzyme-catalyzed H2O2 production (Du and Clemetson, 2002). Fig.6shows the results of assays carried out to study the effect ofHTP1 on the viability ofL. (L.)amazonensispromastigotes.Upon incubation with B. jararaca whole venom, 69%viability was obtained, whereas 47.5% viability wasobserved after incubation with HTP1. Inclusion of catalaseabolished the leishmanicidal activity of both crude venomand HTP1. After incubation with H2O2 (as a positive control)71% of promastigotes remained viable. Incubation of cellswith complete HBSS medium produced 75% viability

A

B

P4

P1

P2

P3

P5

P6

P7

P8

1 10 20 35

Volume (mL)

0

0.1

0.2

0.3

0.4

0.5

Abs280

nm

(

)

0

0.02

0.04

0.06

0.08

Abs436nm(---)

P1 P2 P3 P4

P5 P6 P7 P8

C+

Fig. 3. Anion exchange chromatography ofB. jararaca bactericidal fractions

obtained by gel filtration. (A) Elution profile of fraction P1 subjected to anion

exchange chromatography. Gel filtration fraction P1 (see Fig. 2A) was sus-

pended in 20 mM HEPES, pH 8.0, applied to a Mono Q anion exchange

column and eluted with a linear 00.5 M NaCl gradient (in cinder). Flow rate

was 1 mL/min; 1 mL fractions. Elution was monitored at 280 nm (d). Each

fraction was assayed for LAAO activity using L-leucine as substrate; activity

was recorded at 436 nm (- - - - - -). Asterisks (*) indicate active fractions P5

and P6. (B) Bactericidal activity of Mono Q fractions. Each fraction (P1P8)

was assayed at 0.8 mg/mL on wells made on BHI plates inoculated with

S. aureus. B. jararaca venom was used as positive control (C). Plates wereincubated for approx. 7 h at 37 C.

B

C

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Crude

venom

P1 P2 P3 P4 P5 P6

Abs436nm

5 10 15 20

Volume (mL)

0

0.5

1

1.5

2

Abs

280nm

P2

P3

P4

P1

P6

P5

P1 P2 P3

P4 P5 P6

C+

A

Fig. 2. Gel exclusion chromatography of B. jararaca venom and LAAO and

bactericidalactivity of pooledfractions.(A) Representativeelution profileofB.

jararacacrude venom (10 mg protein) chromatography in a Superdex75

HR10/30 column,usingan FPLC system. Elutionbuffer was 0.15 M ammoniumformiate, pH 6.0; flow rate 0.5 mL/min at room temperature. (B) Bactericidal

activity of gel filtration chromatographic fractions. Each fraction (P1P6) was

assayed at 0.8 mg/mL on wells made on BHI plates inoculated with S. aureus.

B. jararacavenomwas used as positive control (C). Plateswere incubatedfor

approx. 7 h at 37 C. (C) LAAO activity of chromatographic fractions

performed as outlined in materials and methods (Section2.5).



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whereas incubation with 1 M TrisHCl did not influencecell viability (Fig. 6). Regarding hemolytic activity, all

bactericidal fractions and the crude B. jararaca venomproduced significant lysis of horse blood. Fig. 7 showsformation of a greenish halo around the wells indicative ofthe occurrence ofb-hemolysis.

3.8. B. jararaca LAAO specific activity towards

amino acid substrates

Table 3presents the results of a study carried out todetermine the specific activity of fraction HTP1 towardsvarious amino acid substrates. Previously, the specificactivity of HTP1 towards oxidation of L-leucine had beenshown (Figs. 3 and 4). Activity was highest for L-leucine(142.5 U/mg) and L-methionine (136.5 U/mg), followed

by L-arginine (82.2 U/mg), L-tryptophan (76.2 U/mg), andL-phenylalanine (58.2 U/mg). Oxidation of L-asparagineand L-serine by HTP1 was poor and L-proline was notoxidized at all.

3.9. Indirect ELISA for potency estimation

of antibothropic antivenom

The potency of five Bothrops antivenoms samples andone pre-immune serum horse as negative control in pro-tecting against lethality in mice and the ELISA antibodytiters against crude venom and LAAO antigens is shown inTable 2 and Fig. 8. Antivenoms protected against lethality ofB. jararaca with ED50 ranging from 1 to 9 mg/mL. A bestcorrelation was found between ELISA titers and neutral-izing of lethal activity (ED50) when using LAAO to coat theplates. Correlation coefficients (r) between ED50and ELISAantibody titers againstB. jararaca crude venom and LAAOwere 0.747 (p< 0.001) and 0.846 (p< 0.001), respectively(Fig. 6A and B).

4. Discussion

4.1. Screening of antimicrobial activity of arthropod

and ophidian venoms

Our original goal was to identify insect, arachnid, orophidian venoms that could be used as primary source ofantibacterial and/or antiprotozoal components suitable forstructure-based drug design. Initially, our work revealedthat venoms from bees, scorpions, and spiders belonging togeneraApis,Tityus,Lycosa, andPhoneutria, respectively, arenot active in inhibiting the growth of the Gram-positivebacteriumS. aureus at the dose tested (0.8 mg/mL). Theseresults agree with previous reports documenting thatantimicrobial peptides derived from scorpions (Opis-tophtalmus carinatusandParabuthus schlechteri)(Moermanet al., 2002) andLycosaspiders preferentially inhibited thegrowth of Gram-negative bacteria (Yan and Adams, 1998).On the other hand,Moerman et al. (2002)have shown thatmelittin, a major peptide of A. mellifera venom, is moreeffective against Gram-positive bacteria (such as S. aureus)in the same concentration range. Our result indicating thebee venom lower or null microbicidal activity is probablyexpected since we used crude A. melliferavenom extracts.Our work demonstrated the antimicrobial potency ofNeotropical ophidian venoms, particularly those from thegenusBothropsas shown before in several reports (Paramoet al., 1998; Rodrigues et al., 2004; Stabeli et al., 2004). Ingeneral, venoms from the Elapidae and Viperidae familiesare the most active against bacteria (Stiles et al., 1991).Noticeably, we found that among all Bothrops venomstested,B. jararacussu and B. jararaca exhibited the highestinhibitory activity againstS. aureus. As it will be discussedlater, antimicrobial activity of snake venoms has beenassociated with their LAAO and/or phospholipase A2activities. Pessatti et al. (1995) reported a higher LAAOactivity of B. neuwiedii venom from the Amazon region(655.97 U/mL) than those ofB. jararaca and B. jararacussufrom southeast Brazil (314.95 and 391.87 U/mL, respec-tively). Similarly,Tan and Ponndurai (1991)found a higher

A

B

2 4 8 12 16

Volume (mL)

0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

280nm(

)

0

0.02

0.04

0.06

0.08

0.1

Abs436nm(----)

HTP1

1 2

3 4

Fig. 4. Affinity chromatography of active fractions obtained from Mono Q

anion exchange columns. (A) Elution profile of pooled fractions P5 and P6 on

HiTrap Heparin columns using an FPLC system. Sample (11 mg) was applied

onto a column equilibrated with 10 mM Mes, 10 mM Tris, 2 mM Benzami-

dine, pH 6.0, and eluted using a linear 00.4 M NaCl gradient (in cinder).

Flow rate was 0.5 mL/min; 1 mL fractions. Elution was monitored at 280 nm

(d). Each fraction was assayed for LAAO activity using L-leucine as substrate;

activity was recorded at 436 nm (- - -). (B) Bactericidal activity of fraction

HTP1 obtained by affinity chromatography. Samples (0.8 mg/mL) were

assayed for activity against S. aureus. 1. B. jararaca crude venom,

2. HTP1 fraction, 3. HTP1 fraction including catalase (0.3 mg/mL), 4. Catalase

(0.3 mg/mL).



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LAAO activity in B. neuwiedii, and also considerable indi-vidual variation in the enzyme levels among B. jararacaspecimens. It is known the considerable influence ofgeography and ontogeny variation in protein content andquality in snake venoms (see for exampleMeier, 1986). Theconsistent lower bactericidal potency detected by us inBothrops venoms (includingB. neuwidii) other thanB. jar-aracussu and B. jararacacould well be related to a combi-

nation of these factors. Regardless of the basis of suchdifferences, both these venoms were active not only onS.aureus but on other Gram-positive (G) and Gram-negative(G) bacteria. For instance, S. typhimurium and P.

aeruginosa(both G) were sensitive toB. jararacussuandB.jararacavenoms, whereasE. coli (G)remained refractoryto venom action, an observation consistent with otherreports on the activity of viperid venoms (see Blaylock,2000and Stiles et al., 1991). Out of these two most activevenoms, B. jararacussu has been studied intensivelyregarding the microbicidal activities of isolated phospho-lipases (Barbosa et al., 2005) and the molecular cloning of

LAAO (Franca et al., 2007). We then choose B. jararaca as themodel Bothrops venom for isolation of biomolecules withantimicrobial properties.

4.2. Purification ofB. jararacamicrobicidal components

Out of the six fractions obtained by gel exclusionchromatography ofB. jararaca venom, the highest molec-ular mass fraction P1 contained most crude venomsmicrobicidal activity.Maria et al. (1998)obtained a similarelution profile for B. jararaca venom, reporting that thefraction equivalent to P1 contained components whichwere lethal to mice upon intraperitoneal injection. To

further resolve fraction P1 microbicidal/antiprotozoalcomponents, we used a combination of anion exchange andaffinity chromatography steps, which rendered activefraction HTP1, which corresponds to approx. 1.1% of thecrude venoms protein content. Such fraction exhibitedboth anti-S. aureus and LAAO activity which is in agreementwith previous findings on the bactericidal activity of LAAOsfrom the genusBothrops(Stabeli et al., 2004).

4.3. Characterization of fraction HTP1 containing

B. jararacaLAAO

After non-reducing SDS-PAGE, HTP1 was shown tocontain three protein bands, which are unlikely isoforms of

207.0 -

92 .0 -

55.0 -

35.0 -

1 2 3 4A B

Fig. 5. Molecular mass and isoform composition ofB. jararacaLAAO. (A) SDS-PAGE of active chromatographic fractions. 1. CrudeB. jararaca venom, 2. Gel filtrationfraction P1 (seeFig. 2), 3. Anion exchange chromatography fractions P5 and P6 (pool) (see Fig. 3), 4. HiTrap Heparin fraction HTP1 (Fig. 4). Samples (2 mg) were

subjected to electrophoresis in 12.5% gels which were subsequently silver-stained. (B) SDS-PAGE (12.5% gel) of a sample of fraction HTP1 (230 mg) subjected to

deglycosylation as described in Section2.7 of materials and methods.

0

20

40

60

80

100

A B C D E F G H I

Celu

larViability(

)

Fig. 6. Characterization of the leishmanicidal activity ofB. jararaca venom.

(A)L. (L.)amazonensispromastigotes in complete DMEM; (B) promastigotes

(4 105 cells/well) in DMEM 5 mM H2O2; (C) promastigotes in DMEM B.

jararaca crude venom (0.8 mg/mL);(D) promastigotesin DMEM 0.8 mg/mL

B. jararaca crude venom (0.8 mg/mL) catalase (0.3 mg/mL); (E) promasti-

gotes in DMEM affinity chromatography fraction HTP1 (0.8 mg/mL); (F)

promastigotes in DMEM affinity chromatography fraction HTP1 (0.8 mg/

mL) catalase (0.3 mg/mL); (G) promastigotas in DMEM TrisHCl 1 M; (H)

promatigotes in complete HBSS medium; (I) promastigotes in HBSS affinity

chromatography fraction HTP1 (0.8 mg/mL). Viability (median of experi-

ments performed in triplicates) is expressed as a percentage of absorbancereadings at 570 nm of control promastigotes suspended in culture media.



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the LAAO protein moiety itself since the venom used forfractionation was a pool of several specimens. Under suchconditions, we expect that individual variations should beminimal. Accordingly, a single, symmetrical peak was

obtained after subjecting HTP1 to reverse phase HPLC (data

Fig. 7. Hemolytic activity of B. jararaca and bactericidal fractions. Samples

(20 mg) were applied onto wells made of TSA agar prepared with horse blood

as indicated in Section2.8. (A) After 6 h of incubation at 37 C; (B) After 18 h

of incubation at 37 C; 1. Crude venom from B. jararaca; 2. Gel filtration

fraction P1; 3. Mono Q column fraction TAP5/6 (pool of P5 and P6); 4. HiTrap

Heparin column fraction HTP1; 5. HTP1 fraction including catalase (0.3 mg/

mL) (For interpretation of the references to colour in this figure legend, the

reader is referred to the web version of this article.)

Table 2

Potency of bothropic antivenoms in protecting against lethality and ELISA

antibody reactivity of theBothrops jararacacrude venom and LAAO.

Antivenom Sample No Lethality(ED50)

mg/mLaELISA reactivity (492 nm)

B. jararacavenom LA AO

1 Control 0 0.190 0.115

2 1 0.250 0.180

3 3 0.220 0.190

4 5 0.350 0.250

5 7 0.890 0.650

6 9 0.720 0.950

a

Amount of antivenom need to protect half of the mice injected with 5LD50ofB. jararacacrude venom.

Table 3

Specific activity ofB. jararacaLAAO towards amino acid substrates.

Amino acid Specific activity (U/mg)a

L-Leucine 142.5

L-Methionine 136.5

L-Arginine 82.3

L-Tryptophan 76.2

L-Phenylalanine 58.2

L-Valine 58.2L-Histidine 54.1

L-Isoleucine 50.1

L-Cysteine 18.0

L-Lysine 16.0

L-Alanine 16.0

L-Threonine 12.0

L-Asparagine 6.0

L-Serine 4.0

L-Proline 0.0

a One unit of enzyme activity is defined as the oxidation of 1 mM

L-Leucine for min.

Fig. 8. Correlation between ELISA antibody level (absorbance at 492 nm)

and in vivo neutralizing potency ofB. jararaca antivenoms. Ninety-six-well

microtiter plates were coated with crudeB. jararaca venom (A) and LAAO (B)

and 16 antivenoms were used at 1:40,000 dilution. Neutralizing potency

was expressed as effective dose 50% (ED50), defined as the amount (mg) of

venom neutralized by 1.0 mL of antivenom (volume of antivenom needed to

prevent death of 50% of the injected mice). All data points are the means oftwo experiments.

http://-/?-http://-/?-http://-/?-http://-/?-


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not shown), indicating protein homogeneity. Curti et al.(1968)working on the inactivation process of LAAO fromCrotalus adamanteusalso found three protein bands for theactive and inactive forms of the enzyme. Hayes and Wellner(1969), also working on the enzyme from C. adamanteus,found three bands corresponding to enzymatically activeLAAO variants; each of the these bands rendered 57components when subjected to isoelectric focusing. SinceLAAOs are FAD-dependent glycoproteins, its carbohydratemoieties could contribute to differential electrophoreticmigration. Such carbohydrate moieties play a functionalrole in the LAAO-induced cytoxicity, as shown in the case ofC. rhodostoma (Ande et al., 2006), although glycosylationhas shown to be homogenous in this case (Geyer et al.,2001). To explore such possibility, we decided to subjectHTP1 to digestion with Peptide:N-Glycosidase F, anamidase that cleaves between the innermost GlcNAc andasparagine residues of high mannose, hybrid, and complexoligosaccharides from N-linked glycoproteins (Maley et al.,1989). Sugar removal of HTP1 by PNGase F resulted in theappearance of a single band (38.2 kDa) in non-reducingSDS-PAGE, suggesting thatB. jararacaLAAO heterogeneityresides in the glycan moiety. We are currently investigatingthe nature of such moieties and its functional implications.Further proof of the identity of LAAO as the microbicidalenzyme contained in HTP1 relies on its substrate speci-ficity. Kinetic studies indicate thatB. jararacaLAAO is activeagainst hydrophobic amino acids, such as L-Phe, L-Tyr, L-Leu,L-Ile and L-Trp. It is accordance with the substrate specificityof LAAOs fromC. rhodostoma, Naja naja kaouthia (Tan andSwaminathan, 1992), and B. moojeni (Stabeli et al., 2007)venoms. Also, these results agree with the findings ofPessatti et al. (1995)who determined thatL-Met and L-Leuwere the substrates oxidized at a higher rate by LAAO fromBothrops cotiara. B. jararaca LAAO isolated by us differsconsiderably from other characterized ophidian oxidaseson the basis of its molecular mass (38.2 kDa). LAAO fromCrotalus atrox possesses a mass of 60 kDa (Masuda et al.,1997), whereasStabeli et al. (2004) reported a molecularmass of 66 kDa for the enzyme obtained fromB. alternatus.For B. moojeni, Stabeli et al. (2007) reported a mass of64.8 kDa. In general, ophidian LAAOs are homodimers (5070 kDa subunit mass) associated non-covalently (Du andClemetson, 2002). Since a single type of venom maycontain more than one LAAO (Stiles et al., 1991), furtherexperiments are needed to determine whether the enzymewe isolated fromB. jararacavenom is an atypical ophidianLAAO. In this sense, molecular cloning or N-terminalsequencing should be carried out to compare the primarystructures of the B. jararaca enzyme with those isolated andsequenced from the genusBothrops.

4.4. Hydrogen peroxide-mediated antiparasitic

and hemolytic activity ofB. jararacaLAAO

Both B. jararacacrude venom and fraction HTP1 (char-acterized as containing a single 38.2 kDa component)showed activity against L. (L.) amazonensis promastigotesand promoted partial lysis of horse erythrocytes. Previousreports have indicated that B. jararaca venom inhibitedgrowth ofT. cruziand ofL. majorpromastigotes (Gonalves

et al., 2002). Also,Tempone et al. (2001)have verified theleishmanicidal potency ofB. moojenivenom and associatedsuch activity to an LAAO enzyme with a molecular mass of69 kDa (through SDS-PAGE) and 140 kDa by gel filtrationchromatography. The anti-Leishmania activity of fractionHTP1 is dependent on the free amino acid content of theculture media since venom effect on promastigotes grownin complete HBSS medium (lacking amino acids) was null.Therefore, the B. jararaca venom leishmanicidal action isprobably exerted through its LAAO activity given thesubstrate dependence of such effect.

The hemolytic properties of ophidian LAAOs have beenlittle explored. Detected hemolytic activity in the crudevenom ofEristocophis macmahoniand its purified LAAO onsheep eryhrocytes. Ours is the first report of such activityassociated to an LAAO isolated from the genus Bothrops andis most probably related to its H2O2 producing activity.Addition of catalase abolished the microbicidal, leishma-nicidal, and hemolytic activities of fraction HTP1 suggestingthat H2O2, a product of the LAAO-mediated reaction, isinvolved in such effects. This result is in accordance withthe findings of Tempone et al. (2001) who were able tosuppress the leishmanicidal activity ofB. moojeni venomupon incubation with catalase.Leishmania promastigoteslack production of catalase and superoxide dismutase whichrenders parasites extremely sensitive to H2O2produced bymacrophages (Tempone et al., 2001). The enzymes OHAP-1(Trimeresurus flavoviridis) and apoxin I (C. atrox) lost itsapoptotic capacity upon addition of antioxidants such ascatalase and reduced glutathione (Sun et al., 2003; Toriiet al., 1997). Wei et al. (2002) also observed that the plateletaggregation-mediated activity of LAAO isolated fromTrimeresurusmucrosquamatus was also lost upon incubationwith catalase. These results provide further support for ourproposal that the enzyme we have isolated fromB. jararacavenom is an L-amino acid oxidase acting as an indirectmicrobicidal/leishmanicidal agent which promotes celldeath through the oxidizing action of H2O2 on biologicalmembranes. Minimal inhibitory concentration determina-tions and in vivo assayswill be needed in the futureto assessthe possibility of utilizing B. jararaca LAAO as an antimi-crobial and/or leishmanicidal reagent. According to Sorg(2004), microorganism in general are several fold moresensitive to reactive oxygen species (ROS) than humantissues, implying that there is a bactericidal window whereROSconcentrations sufficient to abolish bacterial growth areharmless to the host cells.

4.5. In vitrotechnique for estimating the

potency antibothropic antivenoms

Since the LAAOs have different physiological effects(Wei et al., 2007) and a possible role in the functionaltoxicity of snakebites (e.g. inhibition of platelet aggrega-tion;Tonismagi et al., 2006), we explored the possibility ofdeveloping an enzyme-linked immunosorbent assay(ELISA) to test the potency of antibothropic antivenomsbased on the reactivity towards LAAO. We have previouslyshown that ELISA utilizing the toxic, highest molecularweight gel filtration fraction of B. jararaca venom can beused as an in vitrotechnique for estimating the potency of



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antibothropic antivenoms (Maria et al., 1998). In this study,purified LAAO was used to coat microtiter plates as antigenon the indirect ELISA type and a good correlation wasobserved in entire antibody titers and neutralization of thevenom lethal activity. Our results indicate that this kind ofELISA should be adequate to follow antibody titters duringimmunization procedures as well as during different stagesof the plasma fractionating process and the application ofthe in vivo assay to be restricted due that, this procedurerequires the use and sacrifice of a large number of mice,unacceptable in many countries due to new and rapidlyevolving legislation prohibiting the production of pain andsuffering in the animal involved.

As conclusion our finding showed that LAAO from theB. jararaca venom plays important pharmacological andantimicrobial properties and this protein may involve insome pharmacological effect of the whole venom and alsotheir possible biotechnological use as molecular marker forin vitro stimation of neutralizing potency of horse anti-bothropic antivenoms.

Acknowledgements

This research was supported by the Conselho Nacionalde Desenvolvimento Cientfico e Tecnologico (CNPq) andFundaao de Amparo a Pesquisa do Estado de Minas Gerais(FAPEMIG), Brazil.

Conflict of interest

The authors declare that there is no conflict of interest.

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antigenic, microbicidal and antiparasitic properties of an l-amino

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