RESUMO
Chitin (ß-1,4-linked-N-acetylglucosamine) provides structural integrity to the nematode eggshell and pharyngeal lining. Chitin is synthesized in nematodes, but not in plants and vertebrates, which are often hosts to parasitic roundworms; hence, the chitin metabolism pathway is considered a potential target for selective interventions. Polysaccharide deacetylases (PDAs), including those that convert chitin to chitosan, have been previously demonstrated in protists, fungi and insects. We show that genes encoding PDAs are distributed throughout the phylum Nematoda, with the two paralogs F48E3.8 and C54G7.3 found in C. elegans. We confirm that the genes are somatically expressed and show that RNAi knockdown of these genes retards C. elegans development. Additionally, we show that proteins from the nematode deacetylate chitin in vitro, we quantify the substrate available in vivo as targets of these enzymes, and we show that Eosin Y (which specifically stains chitosan in fungal cells walls) stains the C. elegans pharynx. Our results suggest that one function of PDAs in nematodes may be deacetylation of the chitinous pharyngeal lining.
Assuntos
Amidoidrolases/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/crescimento & desenvolvimento , Faringe/enzimologia , Faringe/crescimento & desenvolvimento , Acetilação , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Quitina , Quitosana/metabolismo , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Faringe/citologia , Filogenia , Estrutura Terciária de Proteína , Interferência de RNA , Alinhamento de Sequência , Solubilidade , Fatores de Tempo , Extratos de TecidosAssuntos
Quitina Sintase/química , Quitina Sintase/metabolismo , Dirofilaria immitis/embriologia , Dirofilaria immitis/metabolismo , Óvulo/metabolismo , Sequência de Aminoácidos , Animais , Quitina Sintase/genética , DNA de Helmintos/análise , Feminino , Masculino , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
BACKGROUND: Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1) and cathepsin Z (Ce-cpz-1) has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting. METHODS AND FINDINGS: RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages. CONCLUSIONS: Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.