Molecular & Biochemical Parasitology 143 (2005) 209–215 Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth Jason M. Correnti a , Paul J. Brindley b , Edward J. Pearce a,∗ a Department of Pathobiology, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA 19104-6008, USA b Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University Health Sciences Center, New Orleans, LA 70112, USA Received 17 March 2005; received in revised form 22 June 2005; accepted 22 June 2005 Available online 13 July 2005 Abstract Schistosoma mansoni is an important flatworm parasite of man that has remained intractable to experimental analyses of gene function. We have developed an approach for using dsRNA to target schistosome transcripts for RNA interference, and used it to address the role of cathepsin B (SmCB1), a cysteine protease that has been proposed to play a central role in hemoglobin digestion in the schistosome gut. Electroporation of 3 h old larval schistosomes with SmCB1-specific dsRNA (SmCB1-dsRNA) resulted in a greater than 10-fold reduction in SmCB1 transcript levels that persisted for >20 days. RNAi mediated reductions in transcript levels led to associated reductions in SmCB1 enzyme activity. Schistosomes treated with SmCB1-dsRNA were viable and developed intestinal heme pigmentation indicative of hemoglobin digestion, but showed significant growth retardation when compared to control parasites, indicating that SmCB1 function is not essential for hemoglobin digestion but is necessary for normal parasite growth. This effect on growth was apparent when parasites were maintained in culture or introduced into mammalian hosts. The report sheds new light on the role of SmCB1 and provides a template for using RNAi to examine gene function in the mammal-parasitic stages of schistosomes during early development in vitro and in vivo. © 2005 Elsevier B.V. All rights reserved. Keywords: Parasite; Helminth; RNAi; Cysteine protease 1. Introduction The blood fluke Schistosoma mansoni and related platyhelminths infect over 200 million people, and are a significant cause of serious disease, especially in the poorer endemic countries where treatment options are limited [1]. While major advances in our understanding of the schistosome genome have occurred recently [2,3], research into the biology of these medically important organisms has been limited due to a lack of robust techniques for the manipulation of schistosome gene expression. Nevertheless, despite these restrictions, significant gains have been made Abbreviations: SmCB1, Schistosoma mansoni cathepsin; L, luciferase; dsRNA, double strand RNA; RNAi, RNA interference ∗ Corresponding author. Tel.: +1 215 573 3493; fax: +1 215 573 7023. E-mail address: ejpearce@mail.med.upenn.edu (E.J. Pearce). in understanding specific areas of schistosome biology. One such example, which also provides prospects for parasite control, is in the understanding of the digestion of hemoglobin, which schistosomes use as a major source of amino acids [4–6]. Schistosomes live intravascularly in their mammalian hosts, where they eat erythrocytes, and it is the oxidation of heme released when globin is digested that results in the formation of the classic black pigment observable within the schistosome gut. Focused examination of the enzymes involved in hemoglobin digestion has implicated a multi-enzyme cascade in which cathepsin B (SmCB1), the most abundant cysteine protease activity found in schistosome extracts, and an enzyme that has demonstrable hemoglobinase activity in vitro, is considered to play a central role [6–8]. Consistent with this, delayed gut development and lethality were observed in schistosomula cultured in the presence of a synthetic peptide inhibitor that 0166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2005.06.007