title: RNA interference (RNAi) for the silencing of extracellular serine proteases genes in Acanthamoeba: molecular analysis and effect on pathogenecity.
abstract: Silencing of extracellular serine protease genes was undertaken by interference RNA (RNAi). Chemically synthesized, small interfering RNA (siRNA) were highly specific and efficient in silencing the catalytic domain of extracellular serine proteases of Acanthamoeba. In order to confirm the silencing phenomenon, the extracellular serine protease activities in RNAi-treated parasites were compared to non-treated parasites, using zymography profiles, Acanthamoeba-conditioned medium (ACM) protease activity, cytotoxicity assays and extracellular serine protease mRNA levels analysis. Zymography profiles showed a decrease in the extracellular protease levels in the moderate pathogenic and pathogenic strains, after treatment with siRNA. These results were supported after the ACM protease activity and CPE assays were performed in all studied isolates, showing a lower protease activity or cytotoxicity both in the pathogenic and moderate pathogenic strains treated with RNAi. These results support that extracellular serine proteases are directly involved in the pathogenesis and virulence of Acanthamoeba.
authors.affiliation: Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, University of La Laguna, Avda. Astrofísico Fco. Sánchez, S/N 38203 La Laguna, Canary Islands, Spain.
authors.forename: Jacob
authors.initials: J
authors.lastname: Lorenzo-Morales
authors.forename: Antonio
authors.initials: A
authors.lastname: Ortega-Rivas
authors.forename: Pilar
authors.initials: P
authors.lastname: Foronda
authors.forename: Néstor
authors.initials: N
authors.lastname: Abreu-Acosta
authors.forename: David
authors.initials: D
authors.lastname: Ballart
authors.forename: Enrique
authors.initials: E
authors.lastname: Martínez
authors.forename: Basilio
authors.initials: B
authors.lastname: Valladares
mesh_terms: D000048:Acanthamoeba / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity*; D000818:Animals; D004794:Enzyme Repression; D017125:Genes, Protozoan; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014774:Virulence
chemical_list: D012333:RNA, Messenger; D016053:RNA, Protozoan; D034741:RNA, Small Interfering; D012697:Serine Endopeptidases