RESUMO
Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (-) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (-) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.
Assuntos
Cisteína Proteases/metabolismo , Ferro/administração & dosagem , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/efeitos dos fármacos , Vagina/parasitologia , Secreções Corporais/parasitologia , Feminino , Humanos , Trichomonas vaginalis/enzimologiaRESUMO
Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (pâ¯<â¯0.05) on cathepsin K, cathepsin B, IL-1ß, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (pâ¯<â¯0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.
Assuntos
Antígenos de Diferenciação/biossíntese , Citrus/química , Cistatinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Animais , Cistatinas/química , Humanos , Macrófagos/patologia , Masculino , Camundongos , Proteínas de Plantas/química , Células RAW 264.7 , Ratos , Ratos WistarRESUMO
Trichomonas vaginalis induces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose on T. vaginalis cytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface of T. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.
Assuntos
Apoptose , Cisteína Endopeptidases/metabolismo , Glucose/metabolismo , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/patogenicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Glucose/farmacologia , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Fenômenos Fisiológicos da Nutrição , Proteólise , Proteínas de Protozoários/metabolismoRESUMO
Piroplasmid parasites comprising of Babesia, Theileria, and Cytauxzoon are transmitted by ticks to farm and pet animals and have a significant impact on livestock industries and animal health in tropical and subtropical regions worldwide. In addition, diverse Babesia spp. infect humans as opportunistic hosts. Molecular phylogeny has demonstrated at least six piroplasmid lineages exemplified by B. microti, B. duncani, C. felis, T. equi, Theileria sensu stricto (T. annulata, T. parva, and T. orientalis) and Babesia sensu stricto (B. bovis, B. bigemina, and B. ovis). C1A cysteine-proteinases (C1A-Cp) are papain-like enzymes implicated in pathogenic and vital steps of the parasite life cycle such as nutrition and host cell egress. An expansion of C1A-Cp of T. annulata and T. parva with respect to B. bovis and B. ovis was previously described. In the present work, C1A-Cp paralogs were identified in available genomes of species pertaining to each piroplasmid lineage. Phylogenetic analysis revealed eight C1A-Cp groups. The profile of C1A-Cp paralogs across these groups corroborates and defines the existence of six piroplasmid lineages. C. felis, T. equi and Theileria s.s. each showed characteristic expansions into extensive families of C1A-Cp paralogs in two of the eight groups. Underlying gene duplications have occurred as independent unique evolutionary events that allow distinguishing these three piroplasmid lineages. We hypothesize that C1A-Cp paralog families may be associated with the advent of the schizont stage. Differences in the invertebrate tick host specificity and/or mode of transmission in piroplasmid lineages might also be associated with the observed C1A-Cp paralog profiles.
RESUMO
Background: Fascioliasis is an infectious disease caused by parasites Fasciola hepatica and F. gigantica. Humans are infected by the consumption of vegetables and water contaminated with the infective form of the parasite. Materials and Methods: In this study, an IgM-ELISA with the cysteine proteinase Fas2 antigen was evaluated with sera from 76 patients infected with F. hepatica, 24 patients with other parasite infections and 84 healthy volunteers. Results: IgM-ELISA resulted in 43% positives in F. hepatica patients with positive serology to Fas2-ELISA, but no positives resulted from testing healthy volunteers and individuals infected with other parasites. The IgM-ELISA diagnostic parameters showed a sensitivity of 43.4% (95% CI 0.321-0.553), specificity of 100% (95% CI 0.957-1), and no cross-reactivity with other parasitic infection. Interference by rheumatoid factor in the IgM immunoassay was controlled by treating sera with rheumatoid factor absorbent before testing. Conclusions: Fas2 antigen is detected by circulating IgM in patients infected with F. hepatica and IgM-ELISA using Fas2 appears as a specific immunoassay to detect the acute phase of the acute phase of F. hepatica infection in humans.
Assuntos
Anticorpos Anti-Idiotípicos/sangue , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/sangue , Cisteína Endopeptidases/sangue , Fasciola hepatica/imunologia , Fasciolíase/sangue , Imunoglobulina G/sangue , Imunoglobulina M/imunologia , Animais , Área Sob a Curva , Ensaio de Imunoadsorção Enzimática , Fasciola hepatica/enzimologia , Fasciolíase/imunologia , Humanos , Fatores Imunológicos/farmacologia , Peru , Valor Preditivo dos Testes , Curva ROC , Fator Reumatoide/farmacologia , Sensibilidade e Especificidade , Estudos SoroepidemiológicosRESUMO
The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter. The active TvLEGU-1 recombinant protein (rTvLEGU-1) was secreted into the medium when tvlegu-1-opt was fused to the Aspergillus niger alpha-amylase signal peptide. The rTvLEGU-1 secretion was influenced by the gene copy number and induction temperature. Data indicate that increasing tvlegu-1-opt gene copy number was detrimental for heterologous expression of the enzymatically active TvLEGU-1. Indeed, expression of TvLEGU-1 had a greater impact on cell viability for those clones with 26 or 29 gene copy number, and cell lysis was observed when the induction was carried out at 30 °C. The enzyme activity in the medium was higher when the induction was carried out at 16 °C and in P. pastoris clones with lower gene copy number. The results presented here suggest that both copy number and induction temperature affect the rTvLEGU-1 expression in its native-like and active conformation.
Assuntos
Cisteína Proteases , Expressão Gênica , Pichia/metabolismo , Proteínas de Protozoários , Proteínas Recombinantes de Fusão , Trichomonas vaginalis/genética , Aspergillus niger/enzimologia , Aspergillus niger/genética , Cisteína Proteases/biossíntese , Cisteína Proteases/química , Cisteína Proteases/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Pichia/genética , Sinais Direcionadores de Proteínas/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Trichomonas vaginalis/enzimologia , alfa-Amilases/biossíntese , alfa-Amilases/química , alfa-Amilases/genéticaRESUMO
Leishmania (Viannia) braziliensis presents adaptive protease-dependent mechanisms, as cysteine proteinases B (CPB). This study investigates the expression of three cpb gene isoforms and CPB enzymatic activity during the parasite differentiation. Relative expression levels of LbrM.08.0810 gene were assessed, exhibiting a higher quantity of transcripts in the logarithmic promastigotes phase than in the stationary promastigotes phase (>1.5 times). The cbp gene tends to decrease during acid pH shock and increases when the temperature rises (>1.3 times). LbrM.08.0820 and LbrM.08.0830 genes exhibited similar expression profiles to LbrM.08.0810 gene, with lower levels being observed overall. The proteolytic activity exhibits a gradual increase during the parasite's differentiation with low levels in samples of logarithmic promastigotes phase (3.2 ± 0.08 mmol min-1 mg protein-1) to a peak of activity after 72 h of incubation at 32 °C (4.2 ± 0.026 mmol min-1 mg protein-1) followed by a subsequent decrease of 68 % of peak activity levels after 96 h of incubation at 32 °C (2.8 ± 0.37 mmol min-1 mg protein-1). These activities were also measured in the presence of selective inhibitors for cysteine proteinases, such as Z-Phe-Phe-fluoromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, demonstrating their source as cathepsin-like proteinases. To the best of our knowledge, this report presents the first description of a modulation of cathepsin L-like expression during the L. (V.) braziliensis in vitro differentiation induced by acid pH and high temperature.
Assuntos
Catepsinas/biossíntese , Diferenciação Celular/efeitos dos fármacos , Cisteína Proteases/biossíntese , Leishmania braziliensis/enzimologia , Animais , Catepsinas/genética , Catepsinas/metabolismo , Diferenciação Celular/genética , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Leishmania braziliensis/crescimento & desenvolvimento , Proteólise/efeitos dos fármacos , TemperaturaRESUMO
Abstract Prior studies demonstrate that a proteinase fraction from Vasconcellea cundinamarcensis V.M. Badillo, Caricaceae, exhibits wound healing activity in gastric and cutaneous models and antitumoral/antimetastatic effects. Here, we present the toxicity, pharmacokinetics and biodistribution data for this proteinase fraction following a single dose into Swiss mice by i.v., s.c. or p.o. routes. The i.v. and s.c. toxicity assays demonstrate that proteinase fraction at ≤20 mg/kg is non-lethal after single injection, while parental administration (p.o.) of ≤300 mg/kg does not cause death. Based on p.o. acute toxicity dose using Organisation for Economic Cooperation and Development protocols, proteinase fraction ranks as Class IV “harmful” substance. Proteinase fraction shows high uptake determined as Kp (distribution tissue/blood) in organs linked to metabolism and excretion. Also, high bioavailability (≈100%) was observed by s.c. administration. The blood contents following i.v. dose fits into a pharmacokinetic bi-compartmental model, consisting of high removal constants – kel 0.22 h−1 and kd 2.32 h−1and a half-life – t½ = 3.13 h. The Ames test of proteinase fraction (0.01–1%) demonstrates absence of mutagenic activity. Likewise, genotoxic evaluation of proteinase fraction (5 or 10 mg/kg, i.p.) shows no influence in micronuclei frequency. In conclusion, the acute doses for proteinase fraction lack mutagenic and genotoxic activity, clearing the way for clinical assays.
RESUMO
OBJECTIVES: The aim of this study was to extend our knowledge about the mechanism involved in the gastroprotective effect of P1G10, a proteolytic fraction rich in cysteine proteinases from Vasconcellea cundinamarcensis (syn. Carica candamarcensis) latex, which demonstrated gastric healing and protection activities in rats. METHODS: Wistar rats were submitted to gastric lesions by indomethacin and treated with P1G10 (10 mg/kg). Free thiol groups and prostaglandin E2 content were measured in gastric mucosal and gastrin levels in blood samples. To evaluate the participation of nitric oxide (NO) or proteolytic activity of P1G10 on its gastroprotective effect, animals were treated with an inhibitor of NO production (L-NAME) or the fraction inhibited by iodoacetamide, respectively. Gastric secretion study (acidity and pepsin activity) was also performed. KEY FINDINGS: P1G10 (10 mg/kg) inhibited the occurrence of gastric lesions by indomethacin, restored the free thiol groups content on gastric mucosa and increased moderately prostaglandin E2 levels (34%). Furthermore, the treatment decreased the gastrin levels (95%), suggesting a possible modulation of secretory activity. This effect was accordant with attenuation of gastric acidity (42%) and pepsin activity (69%) seen in animals subjected to pyloric ligation. The inhibition of NO production or the proteolytic activity of P1G10 does not affect the gastroprotective effect. CONCLUSIONS: These results can explain the gastroprotective activity of P1G10 and serve a basis for further studies of this active principle.
Assuntos
Carica , Cisteína Proteases/farmacologia , Dinoprostona/metabolismo , Ácido Gástrico/metabolismo , Extratos Vegetais/farmacologia , Compostos de Sulfidrila/metabolismo , Animais , Feminino , Ácido Gástrico/química , Ácido Gástrico/fisiologia , Mucosa Gástrica , Gastrinas/biossíntese , Gastrinas/sangue , Indometacina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
The causal agent of trichomoniasis is a parasitic protist, Trichomonas vaginalis, which is rich in proteolytic activity, primarily carried out by cysteine proteases (CPs). Some CPs are known virulence factors. T. vaginalis also possesses three genes encoding endogenous cystatin-like CP inhibitors. The aim of this study was to identify and characterize one of these CP inhibitors. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), a cystatin-like peptidase inhibitor dubbed Trichocystatin-2 (TC-2) was identified in the T. vaginalis active degradome in association with TvCP39, a 39kDa CP involved in cytotoxicity. To characterize the TC-2 inhibitor, we cloned and expressed the tvicp-2 gene, purified the recombinant protein (TC-2r), and produced a specific polyclonal antibody (α-TC-2r). This antibody recognized a 10kDa protein band by western blotting. An indirect immunofluorescence assay (IFA) and cell fractionation assays using the α-TC-2r antibody showed that TC-2 was localized in the cytoplasm and lysosomes and that it colocalized with TvCP39. TC-2r showed inhibitory activity against papain, cathepsin-L, and TvCP39 in trichomonad extracts and live parasites but not legumain-like CPs. Live trichomonads treated with TC-2r showed reduced trichomonal cytotoxicity to HeLa cell monolayers in a TC-2r-concentration-dependent manner. In this study, we identified and characterized an endogenous cystatin-like inhibitor in T. vaginalis, TC-2, which is associated with TvCP39 and appears to regulate the cellular damage caused by T. vaginalis.
Assuntos
Cistatinas/farmacologia , Cisteína Proteases/química , Inibidores de Proteases/farmacologia , Tricomoníase/tratamento farmacológico , Trichomonas vaginalis/enzimologia , Animais , Apoptose , Sequência de Bases , Western Blotting , Catepsina L/antagonistas & inibidores , Proliferação de Células , Células Cultivadas , Clonagem Molecular , Cistatinas/genética , Cistatinas/imunologia , Cisteína Endopeptidases/química , Cisteína Proteases/metabolismo , Citoplasma/enzimologia , Eletroforese em Gel Bidimensional , Células HeLa , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Lisossomos/enzimologia , Masculino , Dados de Sequência Molecular , Filogenia , Inibidores de Proteases/imunologia , RNA Mensageiro/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Tricomoníase/metabolismo , Tricomoníase/microbiologia , Trichomonas vaginalis/genéticaRESUMO
Trichomonas vaginalis has multiple proteinases, mainly of the cysteine type (CPs), including a 34 kDa precursor cathepsin L-like CP dubbed TvCP4. TvCP4 is an iron-up-regulated CP. The goal of this work was to identify the role of TvCP4 in the virulence of T. vaginalis. We cloned, expressed, and purified the recombinant mature enzyme region of TvCP4 (TvCP4r) to produce a rabbit polyclonal antibody (α-TvCP4r). This antibody reacted with a â¼24 kDa protein band in total protein extracts that could correspond to the mature enzyme. By two-dimensional western blot assays TvCP4 corresponded to three protein spots of â¼24 kDa with pI values of â¼6.7, 6.9, and 7.0 and two spots of â¼22 and â¼21 kDa with a pI of 6.9, as confirmed by mass spectrometry. As expected, a higher amount of TvCP4 was detected in cytoplasmic vesicles, lysosomes, and on the surface of iron-rich parasites when compared with normal and iron-depleted parasites. The α-TvCP4r antibody protected human erythrocytes from trichomonal lysis. Additionally, TvCP4 is expressed during infection and is part of the released products detected in vaginal fluids of patients with trichomonosis. Thus, data show that TvCP4 is an iron-induced CP that participates in T. vaginalis haemolysis.
Assuntos
Proteínas de Bactérias/metabolismo , Cisteína Proteases/metabolismo , Hemólise , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/fisiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína Proteases/química , Cisteína Proteases/genética , Eritrócitos/parasitologia , Humanos , Ponto Isoelétrico , Espectrometria de Massas , Peso Molecular , Trichomonas vaginalis/patogenicidade , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The development of resistance to anthelmintics has prompted research into alternative methods of controlling intestinal nematodes in ruminants. This study aimed to assess the activity of Ananas comosus on Haemonchus contortus in Santa Inês sheep. The aqueous extract of pineapple skin (AEPS), bromelain from pineapple stems (B4882) and residue from pineapple processing was evaluated in in vitro and in vivo tests. The enzymatic activity of substances was analyzed by the azocasein method. The egg hatch test (EHT) and larval development test (LDT) were performed using the Embrapa2010 isolate of H. contortus. In the in vivo test, 36 sheep artificially infected with H. contortus were divided into six groups: G1: 2 g/kg BW of the aqueous extract administered for three days; G2: 2 g/kg BW of the industrial pineapple residue for 60 days; G3: 180 mg/animal of bromelain in a single dose; G4: negative control I; G5: positive control (levamisole phosphate); and G6: negative control II. The eggs per gram (EPG) in the feces were counted till 28 days after treatment. LC50 and LC90 were obtained by the probit procedure, while the in vivo test results were analyzed by GLM. The aqueous extract in the in vitro and in vivo test, the bromelain and industrial residue presented 0.102, 0.157, 1.864 and 0.048 enzyme units/mL, respectively. In the egg hatch test, the LC50 and LC90 were respectively 31 and 81 mg/mL for the aqueous extract and 0.50 and 2 mg/mL for bromelain. In the larval development test, the LC50 and LC90 were respectively 1.7 and 7.3 mg/mL for the aqueous extract and 0.019 and 0.086 mg/mL for bromelain. In the in vivo test, the general efficacies of the treatments in relation to the negative control were 22.6%, 42.2%, 3.65% and 89% for the aqueous extract, industrial pineapple residue, bromelain and positive control respectively. The transformed EPG values were 3.19 ± 0.59, 3.32 ± 0.25, 2.85 ± 0.66, 3.44 ± 0.50, 2.28 ± 0.93 and 2.75 ± 0.94 for the aqueous extract, industrial residue, bromelain, negative control I, positive control and negative control II respectively. The results for all the treated groups differed significantly (p<0.05) from the positive control, and although the residue presented efficacy of 42.2%, there was no statistical difference (p>0.05) in relation to the negative control. Therefore, both the aqueous extract and bromelain were effective in vitro, but showed reduced anthelmintic efficacy in vivo. For the pineapple residue, the 42.2% in vivo efficacy in reducing the EPG and the possibility of reducing environmental contamination through reuse of industrial residue indicate it can also be useful for control of this parasite.
Assuntos
Ananas/química , Hemoncose/veterinária , Haemonchus , Extratos Vegetais/uso terapêutico , Doenças dos Ovinos/parasitologia , Animais , Hemoncose/tratamento farmacológico , Hemoncose/parasitologia , Larva , Extratos Vegetais/química , Ovinos , Doenças dos Ovinos/tratamento farmacológicoRESUMO
It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.