Assuntos
Doenças Transmitidas por Alimentos/parasitologia , Doenças Parasitárias/parasitologia , Animais , Anisaquíase/epidemiologia , Anisaquíase/parasitologia , Anisakis/isolamento & purificação , Anisakis/patogenicidade , Comportamento Alimentar , Doenças Transmitidas por Alimentos/epidemiologia , Humanos , Japão/epidemiologia , Legislação sobre Alimentos , Paragonimíase/epidemiologia , Paragonimíase/parasitologia , Paragonimus westermani/isolamento & purificação , Paragonimus westermani/patogenicidade , Doenças Parasitárias/epidemiologia , Doenças Parasitárias/prevenção & controle , Fatores de TempoRESUMO
Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.
Assuntos
Sistema Digestório/citologia , Doenças do Cão/patologia , Epitélio/parasitologia , Hemoglobinas/metabolismo , Leucil Aminopeptidase , Paragonimus westermani/patogenicidade , Animais , Clonagem Molecular , DNA Complementar/genética , Sistema Digestório/enzimologia , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Epitélio/enzimologia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Leucil Aminopeptidase/química , Leucil Aminopeptidase/genética , Leucil Aminopeptidase/metabolismo , Masculino , Dados de Sequência Molecular , Paragonimíase/imunologia , Paragonimíase/parasitologia , Paragonimíase/patologia , Paragonimíase/veterinária , Paragonimus westermani/enzimologia , Paragonimus westermani/genética , Paragonimus westermani/crescimento & desenvolvimento , Filogenia , Ratos , Ratos Sprague-Dawley , Análise de Sequência de DNARESUMO
Paragonimus westermani is a trematode parasite, which causes pulmonary and/or extrapulmonary granulomatous disease in humans. Successful invasion of the host tissue is critical for the survival of this tissue-invasive parasite. The enzymatic hydrolysis of host proteins is clearly a prerequisite of this process. In this study, we have investigated the functional roles of the excretory-secretory cysteine proteases of P. westermani newly excysted metacercariae (PwNEM) in tissue invasion. The 27 and 28 kDa enzymes (PwMc27 and PwMc28) purified from PwNEM excretory-secretory products (ESP), preferentially degraded fibrillar proteins, but not globular proteins. PwMc28 significantly facilitated the invasion of PwNEM into mouse peritoneum, whereas a diffusible cysteine protease inhibitor, trans-epoxysuccinyl-L-leuciloamido-(4-guanidino) butane (E-64) inhibited this process dose-dependently. Two distinct isoforms of PwMc28 (PwMc28a and PwMc28b), which exhibited two amino acid differences in their mature domains, were identified by tandem mass spectrometry and sequence analysis. Both enzymes were localized at the tegument on the anterior border and on the oral sucker, which suggests excretion-secretion via exocytosis or via the excretory canal network. The mRNA transcripts of PwMc28a and b were expressed abundantly during the active invasion/migration through the host's tissues, suggesting their relevant function to tissue invasion/migration in the definitive host.