RESUMO
Clonorchis sinensis (C. sinensis), a trematode parasite that invades the hypoxic hepatobiliary tract of vertebrate hosts requires a considerable amount of oxygen for its sexual reproduction and energy metabolism. However, little is known regarding the molecular mechanism of C. sinensis involved in the adaptation to the hypoxic environments. In this study, we investigated the molecular structures and induction patterns of hypoxia-inducible factor-1α (HIF-1α) and other basic helix-loop-helix and Per-Arnt-Sim (bHLH-PAS) domain-containing proteins such as HIF-1ß, single-minded protein and aryl hydrocarbon receptor, which might prompt adaptive response to hypoxia, in C. sinensis. These proteins possessed various bHLH-PAS family-specific domains. Expression of C. sinensis HIF-1α (CsHIF-1α) was highly induced in worms which were either exposed to a hypoxic condition or co-incubated with human cholangiocytes. In addition to oxygen, nitric oxide and nitrite affected the CsHIF-1α expression depending on the surrounding oxygen concentration. Treatment using a prolyl hydroxylase-domain protein inhibitor under 20%-oxygen condition resulted in an increase in the CsHIF-1α level. Conversely, the other bHLH-PAS genes were less responsive to these exogenous stimuli. We suggest that nitrite and nitric oxide, as well as oxygen, coordinately involve in the regulation of HIF-1α expression to adapt to the hypoxic host environments in C. sinensis.
Assuntos
Clonorchis sinensis/genética , Clonorchis sinensis/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonorquíase/complicações , Clonorquíase/parasitologia , Clonorchis sinensis/química , Clonorchis sinensis/classificação , DNA Complementar/química , Expressão Gênica , Sequências Hélice-Alça-Hélice/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Conformação Molecular , Óxido Nítrico/farmacologia , Nitritos/farmacologia , Filogenia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Sprague-DawleyRESUMO
Larval Taeniidae, such as metacestodes of Taenia solium, Echinococcus granulosus, and Echinococcus multilocularis, produce chronic and fatal helminthic diseases. Proper identification of these zoonotic cestodiases is often challenging and is hampered in some clinical settings. Endophilin B1 plays critical roles in the maintenance of membrane contours and endocytosis. We isolated proteins homologous to endophilin B1 from T. solium, Taenia saginata, and Taenia asiatica The three Taeniidae endophilin B1 proteins shared 92.9 to 96.6% sequence identity. They harbored a Bin1/amphiphysin/Rvs (BAR) domain and residues for a dimeric interface but lacked a SRC homology 3 (SH3) domain. Endophilin B1 showed a unique immunological profile and was abundantly expressed in the tegumental syncytium of Taeniidae metacestodes and adults. Bacterially expressed recombinant T. solium endophilin B1 (rTsMEndoB1) demonstrated a sensitivity of 79.7% (345/433 cases) for serodiagnosis of larval Taeniidae infections. The protein showed strong immune recognition patterns against sera from patients with chronic neurocysticercosis, cystic echinococcosis, or advanced-stage alveolar echinococcosis. Adult Taeniidae infections exhibited moderate degrees of positive antibody responses (65.7% [23/35 samples]). rTsMEndoB1 showed some cross-reactivity with sera from patients infected with Diphyllobothriidae (23.6% [25/106 samples]) but not with sera from patients with other parasitic diseases or normal controls. The specificity was 91.7% (256/301 samples). The positive and negative predictive values were 93.6% and 73.4%, respectively. Our results demonstrate that Taeniidae endophilin B1 may be involved in the control of membrane dynamics, thus contributing to shaping and maintaining the tegumental curvature. rTsMEndoB1 may be useful for large-scale screening, as well as for individual diagnosis and follow-up surveillance of Taeniidae infections.
Assuntos
Aciltransferases/biossíntese , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/biossíntese , Perfilação da Expressão Gênica , Imunoensaio/métodos , Taenia/imunologia , Teníase/diagnóstico , Aciltransferases/genética , Aciltransferases/imunologia , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Humanos , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Análise Espaço-TemporalRESUMO
BACKGROUND: Clonorchis sinensis causes a major food-borne helminthic infection. This species locates in mammalian hepatobiliary ducts, where oxidative stressors and hydrophobic substances are profuse. To adapt to the hostile micromilieu and to ensure its long-term survival, the parasite continuously produces a diverse repertoire of antioxidant enzymes including several species of glutathione transferases (GSTs). Helminth GSTs play pertinent roles during sequestration of harmful xenobiotics since most helminths lack the cytochrome P-450 detoxifying enzyme. METHODS: We isolated and analyzed the biochemical properties of two omega-class GSTs of C. sinensis (CsGSTo1 and CsGSTo2). We observed spatiotemporal expression patterns in accordance with the maturation of the worm's reproductive system. Possible biological protective roles of CsGSTos in these organs under oxidative stress were investigated. RESULTS: The full-length cDNAs of CsGSTo1 and 2 constituted 965 bp and 1,061 bp with open reading frames of 737 bp (246 amino acids) and 669 bp (223 amino acids). They harbored characteristic N-terminal thioredoxin-like and C-terminal α-helical domains. A cysteine residue, which constituted omega-class specific active site, and the glutathione-binding amino acids, were recognized in appropriate positions. They shared 44 % sequence identity with each other and 14.8-44.8 % with orthologues/homologues from other organisms. Bacterially expressed recombinant proteins (rCsGSTo1 and 2) exhibited dehydroascorbate reductase (DHAR) and thioltransferase activities. DHAR activity was higher than thioltransferase activity. They showed weak canonical GST activity toward 1-chloro-2,4-dinitrobenzene. S-hexylglutathione potently and competitively inhibited the active-site at nanomolar concentrations (0.63 and 0.58 nM for rCsGSTo1 and 2). Interestingly, rCsGSTos exhibited high enzyme activity toward mu- and theta-class GST specific substrate, 4-nitrobenzyl chloride. Expression of CsGSTo transcripts and proteins increased beginning in 2-week-old juveniles and reached their highest levels in 4-week-old adults. The proteins were mainly expressed in the elements of the reproductive system, such as vitelline follicles, testes, seminal receptacle, sperm and eggs. Oxidative stressors induced upregulated expression of CsGSTos in these organs. Regardless of oxidative stresses, CsGSTos continued to be highly expressed in eggs. CsGSTo1 or 2 overexpressing bacteria demonstrated high resistance under oxidative killing. CONCLUSIONS: CsGSTos might be critically involved in protection of the reproductive system during maturation of C. sinensis worms and in response to oxidative conditions, thereby contributing to maintenance of parasite fecundity.
Assuntos
Clonorchis sinensis/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glutationa Transferase/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Clonorquíase/parasitologia , Glutationa Transferase/classificação , Glutationa Transferase/genética , Estresse Oxidativo , Filogenia , Ratos , Ratos Sprague-Dawley , Reprodução , Fatores de TempoRESUMO
Alveolar echinococcosis (AE), caused by the Echinococcus multilocularis metacestode, represents one of the most frequently fatal zoonoses. Early diagnosis significantly reduces morbidity and mortality associated with AE. Diagnosis of AE largely depends on a combination of imaging and serological tests due to its minimal clinical manifestations. Several antigens derived from the whole worm and protoscolex have been targeted for AE serodiagnosis, while the antigenic properties of E. multilocularis hydatid fluid (EmHF) are unclear. We observed two AE-specific 6- and 8-kDa antigen proteoforms through an immunoproteome array of the EmHF. We identified these proteins as representing an E. multilocularis antigen B3 (EmAgB3) isoform, and the proteins were shown to be encoded by the same gene. We cloned the gene and expressed the recombinant EmAgB3 protein (rEmAgB3) in Escherichia coli. rEmAgB3 exhibited sensitivity of 90.9% (80/88 cases) and specificity of 98.5% (597/606 samples) by immunoblotting. The positive and negative predictive values were 89.9% and 98.6%, respectively. The protein did not show antibody responses to 33 AE sera collected during posttreatment follow-up monitoring. Mouse sera experimentally infected with AE protoscoleces began to demonstrate specific antibody responses to native and recombinant EmAgB3 6 months after infection. At that stage, fully mature metacestode vesicles that harbored the brood capsule, primary cell, and protoscolex were observed within an AE mass(es). The response declined along with worm degeneration. Our results demonstrate that the immune responses to this EmAgB3 isoform were highly correlated with worm viability accompanied with AE progression. rEmAgB3 is a promising biomarker for serological assessment of AE patients.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Equinococose Hepática/diagnóstico , Echinococcus multilocularis/imunologia , Adolescente , Adulto , Idoso , Animais , Criança , Equinococose , Equinococose Hepática/imunologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: Cystic echinococcosis (CE), caused by Echinococcus granulosus metacestode, invokes a serious public health concern. Early diagnosis has great impacts on reduction of disability-adjusted life years. Several antigen B-related molecules (EgAgB; EgAgB1-5) are known to be immunopotent, but detection of EgAgB is variable in many patients and may not allow reliable interpretation of its immunological relevance. More importantly, the immunoproteome profile of hydatid fluid (HF) has not been addressed. METHODS: We conducted a proteome analysis of the HF of a single fertile cyst of CE1 and CE2 stages through two-dimensional electrophoresis (2-DE). Each protein spot was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We subsequently determined the immunoproteome profile employing patient sera of entire disease spectrum from CE1 to CE5 stages. RESULTS: We identified 40 parasite proteins, of which EgAgB (28 spots) and antigen 5 (EgAg5; 5 molecules) were abundant. EgAgB proteoforms constituted the majority, mostly EgAgB1 (24 spots), followed by EgAgB2 and EgAgB4 (2 spots each). EgAgB3 was detected only by liquid chromatography-MS/MS. EgAgB5 was not recognized. We also detected 38 host proteins, which were largely composed of serum components, antioxidant/xenobiotic enzymes, and enzymes involved in carbohydrate metabolism. CE1 and CE2 HF exhibited comparable spotting patterns, but CE2 HF harbored greater amounts of EgAgB and EgAg5 complexes. CE sera demonstrated complicated immune recognition patterns according to the disease progression; CE2 and CE3 stages exhibited strong antibody responses against diverse EgAgB and EgAg5 proteoforms, while CE1, CE4, and CE5 stages mainly reacted to EgAg5 and cathepsin B. Patient sera of alveolar echinococcosis (AE) cross-reacted with diverse EgAgB isoforms (36%). EgAg5 and cathepsin B also demonstrated cross-reactions with sera from neurocysticercosis and sparganosis. CONCLUSIONS: Our results demonstrated that detection of a single defined molecule may not properly diagnose CE, since specific immunodominant epitopes changed as the disease progresses. Immunoproteome analysis combined with imaging studies may be practical in the differential diagnosis of CE from AE and other cystic lesions, as well as for staging CE, which are pertinent to establish appropriate patient management.
Assuntos
Líquido Cístico/química , Equinococose/parasitologia , Echinococcus granulosus/metabolismo , Proteínas de Helminto/metabolismo , Imunoproteínas/metabolismo , Animais , Feminino , Proteínas de Helminto/genética , Humanos , Imunoproteínas/química , Masculino , Isoformas de Proteínas , Proteômica , TranscriptomaRESUMO
OBJECTIVES: Neurocysticercosis (NC), an infection of the central nervous system with Taenia solium metacestodes (TsM), invokes a formidable neurological disease. A bundle of antigens is applicable for serodiagnosis of active cases, while they demonstrate fairly low reactivity against sera of chronic NC. Identification of sensitive biomarkers for chronic NC is critical for appropriate management of patients. METHODS: Proteome analysis revealed several isoforms of 65- and 83-kDa TsM fasciclin-like proteins (TsMFas) to be highly reactive with sera of chronic NC. A cDNA encoding one of the 83-kDa TsMFas (TsMFas1) was isolated from a cDNA library. We expressed a recombinant protein (rTsMFas1) and evaluated its diagnostic potential employing sera from chronic NC (n = 80), tissue-invasive cestodiases (n = 169) and trematodiases (n = 80) and those of normal controls (n = 50). RESULTS: Secretory TsMFas1 was composed of 766 amino acid polypeptide and harboured fasciclin and fasciclin-superfamily domains. The protein was constitutively expressed in metacestode and adult stages, with preferential locality in the scolex. Bacterially expressed rTsMFas1 exhibited 78.8% sensitivity (63/80 cases) and 93% specificity (278/299 samples) in diagnosing chronic NC. Some cross-reactivity was observed with sera of cystic echinococcosis (10/56, 17.8%) and sparganosis (4/50, 8%). Positive and negative predictive values were 75% and 95.5%, respectively. CONCLUSION: TsM fasciclin-like protein may be useful for differential diagnosis of chronic NC in clinical settings, especially where both NC and other infectious cerebral granulomatoses are prevalent.
RESUMO
In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19(+) B cells was observed as early as week 1 post-infection while CD4(+)/CD8(+) T cells were down-regulated. Accumulation of Mac1(+) cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1ß expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-ß were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-ß and IL-4 during the early stages of infection.
Assuntos
Fasciola hepatica/imunologia , Fasciolíase/imunologia , Interleucina-4/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Regulação para Baixo , Imunofenotipagem , Terapia de Imunossupressão , Interleucina-4/sangue , Interleucina-4/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Baço/imunologia , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic ß-barrel composed of 10 anti-parallel ß-strands and two α-helices. TsMFABP2 harbored two additional loops between ß-strands two and three, and ß-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol. CONCLUSIONS/SIGNIFICANCE: The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design.
Assuntos
Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Taenia solium/genética , Sequência de Aminoácidos , Animais , DNA de Helmintos/química , DNA de Helmintos/genética , Proteínas de Ligação a Ácido Graxo/química , Ácidos Graxos/metabolismo , Dados de Sequência Molecular , Peso Molecular , Filogenia , Ligação Proteica , Conformação Proteica , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Diphyllobothrium latum infections in 4 young Korean men detected from 2008 to 2012 are presented. Three were diagnosed based on spontaneously discharged strobila of the adult worm in their feces, and 1 case was diagnosed by finding the worm at colonoscopy examination in a local clinic. The morphologic characteristics of the gravid proglottid and eggs were consistent with D. latum. All patients were treated with praziquantel 15 mg/kg, and follow-up stool examinations were done at 2 months after the medication. The main clinical complaints were intermittent gastrointestinal troubles such as indigestion, abdominal distension, and spontaneous discharge of tapeworm's segments in their feces. The most probable source of infection was the flesh of salmon or trout according to a patient's past history. These are the 45th to 48th recorded cases diagnosed by the adult worm in the Republic of Korea since 1971.
Assuntos
Difilobotríase/diagnóstico , Difilobotríase/patologia , Diphyllobothrium/isolamento & purificação , Adolescente , Adulto , Animais , Anti-Helmínticos/administração & dosagem , Colonoscopia , Difilobotríase/tratamento farmacológico , Difilobotríase/parasitologia , Diphyllobothrium/anatomia & histologia , Fezes/parasitologia , Humanos , Masculino , Microscopia , Praziquantel/administração & dosagem , República da Coreia , Adulto JovemRESUMO
Dendritic cells have been known as a member of strong innate immune cells against infectious organelles. In this study, we evaluated the cytokine expression of splenic dendritic cells in chronic mouse toxoplasmosis by tissue cyst-forming Me49 strain and demonstrated the distribution of lymphoid dendritic cells by fluorescence-activated cell sorter (FACS). Pro-inflammatory cytokines, such as IL-1α, IL-1ß, IL-6, and IL-10 increased rapidly at week 1 post-infection (PI) and peaked at week 3 PI. Serum IL-10 level followed the similar patterns. FACS analysis showed that the number of CD8α(+)/CD11c(+) splenic dendritic cells increased at week 1 and peaked at week 3 PI. In conclusion, mouse splenic dendritic cells showed early and rapid cytokine changes and may have important protective roles in early phases of murine toxoplasmosis.
Assuntos
Citocinas/sangue , Citocinas/metabolismo , Células Dendríticas/imunologia , Baço/imunologia , Toxoplasmose Animal/imunologia , Animais , Antígeno CD11c/análise , Antígenos CD8/análise , Células Dendríticas/química , Modelos Animais de Doenças , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Doenças dos Roedores/imunologia , Fatores de TempoRESUMO
Sparganosis is a tissue invading helminthiasis infecting intermediate hosts, including humans. Strong immune responses are expected to occur in early phases of infection. Thus, we investigated cytokine expressions in splenic dendritic cells and in sera after experimental infection of mice. In splenic dendritic cells, TNF-α and IL-1ß expression peaked at week 1 and week 3 post-infection (PI), respectively, and also early phase (week 2 PI) depressed cytokine expression was noticed. Serum IL-1ß concentration increased significantly at week 2 PI and peaked at week 6 PI, and that of TNF-α peaked at week 6 PI. These results showed that pro-inflammatory cytokines, TNF-α and IL-1ß, are chronologically regulated in mouse sparganosis.
Assuntos
Células Dendríticas/imunologia , Interleucina-1beta/sangue , Interleucina-1beta/metabolismo , Esparganose/imunologia , Baço/imunologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos BALB C , Doenças dos Roedores/imunologia , Soro/químicaRESUMO
Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.
Assuntos
Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/farmacologia , Spirometra/metabolismo , Animais , Cistatinas/farmacologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/químicaRESUMO
After infection of male mice with the plerocercoids (spargana) of Spirometra mansoni, serum levels of estrogen and testicular weight were analyzed by enzyme-linked immunosorbent assay (ELISA) and weighing machine, respectively. The serum level of estrogen increased progressively in infected mice compared with normal controls, whereas the testicular weight of infected mice decreased significantly (P < 0.05). These results suggest that certain substances from spargana change the steroid hormone metabolisms in the host by unknown pathways, and chronic infection may contribute to change of the function of steroid hormone target organ, i.e., testis, in male mice.
Assuntos
Feminização/parasitologia , Esparganose/complicações , Testículo/parasitologia , Animais , Estrogênios/sangue , Masculino , Camundongos , Tamanho do Órgão , Testículo/patologia , Fatores de TempoRESUMO
A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.
Assuntos
Cisteína Endopeptidases/isolamento & purificação , Taenia solium/enzimologia , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Colágeno/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Imunoglobulina G/metabolismo , Ácido Iodoacético/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Soroalbumina Bovina/metabolismoRESUMO
After collecting calcareous corpuscles from plerocercoid of Spirometra mansoni (sparganum), we evaluated the antigenic values of calcareous corpuscles binding proteins obtained from the cyst fluid of Taenia solium metacestodes. Immunoblot analysis revealed that cysticercosis patient sera strongly recognized 10 and 95 kDa calcareous corpuscles binding proteins. This result demonstrated that calcareous corpuscles are bound with major secretory antigenic proteins, which is possibly involved in the secretory pathways of the 10 and 95 kDa proteins presenting in the cyst fluid of T. solium metacestodes.
Assuntos
Antígenos de Helmintos/análise , Proteínas de Transporte/imunologia , Proteínas de Helminto/imunologia , Taenia solium/química , Animais , Cisticercose/diagnóstico , Cisticercose/imunologia , Humanos , Immunoblotting/métodos , Peso Molecular , Testes Sorológicos , Plerocercoide , Taenia solium/imunologiaRESUMO
A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.
Assuntos
Antígenos de Helmintos/análise , Esparganose/parasitologia , Spirometra/metabolismo , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/metabolismo , Carboidratos/análise , Carboidratos/imunologia , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/análise , Epitopos/imunologia , Hexosaminidases/metabolismo , Humanos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Ácido Periódico/química , Plerocercoide/imunologia , Plerocercoide/metabolismo , Spirometra/imunologiaRESUMO
The 150 kDa protein of cyst fluid (CF) of Taenia solium metacestodes was purified by ammonium sulfate fractionation and Superose 6 HR gel filtration chromatography. The purified protein consisted of three subunits (15, 10 and 7 kDa proteins), which were analyzed with the use of a 7.5-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunofluorescence study was carried out by using immunize specific polyclonal antibody. Positive reactions were noticed at bladder walls, calcareous corpuscles, granules of cyst fluid and some host tissue surrounding the bladder wall of the metacestodes. These results suggest that the 150 kDa protein was secreted into host tissues, inducing immune responses in the host, and it may play important roles in the cellular physiology of the parasites.
Assuntos
Líquido Cístico/química , Cisticercose/metabolismo , Proteínas de Helminto/isolamento & purificação , Taenia solium/metabolismo , Animais , Fracionamento Químico , Cromatografia em Gel , Líquido Cístico/metabolismo , Líquido Cístico/parasitologia , Cisticercose/parasitologia , Eletroforese em Gel de Poliacrilamida , Proteínas de Helminto/química , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Peso Molecular , Suínos , Doenças dos Suínos/parasitologiaRESUMO
The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.