Affiliation: | a Laboratório de Microbiologia, Departamento de Análises Clínicas, Universidade Estadual de Maringá, Av. Colombo, 5790, CEP 87030-121, Maringá, PR, Brazil b Laboratório de Biotecnologia Universidade Estadual do Norte Fluminense, CEP 28015-620, Rio de Janeiro, Brazil c Laboratório de Biologia Celular e Tecidual, Universidade Estadual do Norte Fluminense, CEP 28015-620, Rio de Janeiro, Brazil d Laboratory of Medical Entomology, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, CEP 30190-002, Brazil |
Abstract: | Measurement of the hydrolysis of specific fluorogenic substrates by spectrophotometry as well as the substrate activity–SDS–PAGE gel analysis of the chitinolytic activity in Aedes aegypti guts showed that both chitinase and β-N-acetylglucosaminidase are present and physiologically active. Both enzymes were present even in guts from unfed insects, but the activities increased rapidly after feeding on blood or an artificial protein-free diet. Chitinase activity was predominantly of the ‘endo’-type, reaching its maximum activity at 36 h and then declining to very low levels after the degradation of the peritrophic matrix (PM). Chitinase assay in gels after SDS–PAGE was a very sensitive method that allowed us to detect two chitinases with distinct molecular weights in the mosquito gut. Hydrolysis of a chitinase-specific substrate by chitinolytic activities in the mosquito guts was inhibited by allosamidin, a potent chitinase inhibitor. Allosamidin treatment led to the formation of an atypical thick PM, while the addition of exogenous chitinase completely blocked its formation. This chitinolytic system appears to operate both on the formation and degradation of the PM. Since the PM is involved in pathogen invasion, these results are important in facilitating a search for mechanisms that can block pathogen development in the mosquito vector. |