RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting.
We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets.
Lustigman
Sara
S
Laboratory of Molecular Parasitology, Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, NY 10021, USA. slustigman@nybloodcenter.org
Zhang
Jun
J
Liu
Jing
J
Oksov
Yelena
Y
Hashmi
Sarwar
S
D000818:Animals; D001483:Base Sequence; D056657:Cathepsin K; D056668:Cathepsin L; D056663:Cathepsin Z; D002403:Cathepsins / Q000235:genetics / Q000502:physiology*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000502:physiology*; D004794:Enzyme Repression / Q000235:genetics; D017238:Genes, Helminth; D007814:Larva / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D018978:Molting; D017181:Onchocerca volvulus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000648:ultrastructure
D012330:RNA, Double-Stranded; D002403:Cathepsins; C534182:CTSZ protein, human; D056663:Cathepsin Z; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534130:CTSK protein, human; D056657:Cathepsin K