A pore-forming haemolysin from the hookworm, Ancylostoma caninum Tegan A. Don a,b , Malcolm K. Jones a,b , Danielle Smyth a , Peter O’Donoghue b , Peter Hotez c , Alex Loukas a,c,d, * a Division of Infectious Diseases and Immunology, Helminth Biology Laboratory, Queensland Institute of Medical Research, 300 Herston Rd, Brisbane, Qld 4006, Australia b Department of Microbiology and Parasitology, The University of Queensland, Brisbane, Qld 4097, Australia c Department of Microbiology and Tropical Medicine, George Washington University Medical Center, Washington, DC 20037, USA d Australian Centre for International and Tropical Health and Nutrition, The University of Queensland, Brisbane, Qld 4029, Australia Received 5 March 2004; received in revised form 22 April 2004; accepted 25 April 2004 Abstract Hookworms feed on blood, but the mechanism by which they lyse ingested erythrocytes is unknown. Here we show that Ancylostoma caninum, the common dog hookworm, expresses a detergent soluble, haemolytic factor. Activity was identified in both adult and larval stages, was heat-stable and unaffected by the addition of protease inhibitors, metal ions, chelators and reducing agents. Trypsin ablated lysis indicating that the haemolysin is a protein. A closely migrating doublet of hookworm proteins with apparent molecular weights of 60 – 65 kDa bound to the erythrocyte membrane after lysis of cells using both unlabeled and biotinylated detergent-solubilised hookworm extracts. In addition, separation of detergent-soluble parasite extracts using strong cation-exchange chromatography, resulted in purification of 60–65 kDa proteins with trypsin-sensitive haemolytic activity. Erythrocytes lysed with particulate, buffer-insoluble worm extracts were observed using scanning electron microscopy and appeared as red cell ghosts with approximately 100 nm diameter pores formed in the cell membranes. Red blood cell ghosts remained visible indicating that lysis was likely caused by pore formation and followed by osmotic disruption of the cell. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Ancylostoma caninum; Haemolysin; Pore-former; Erythrocyte; Hookworm 1. Introduction The human hookworms, Ancylostoma duodenale and Necator americanus infect almost one billion people (de Silva et al., 2003) and are a major cause of blood loss and iron deficiency anaemia. A related cosmopolitan parasite, Ancylostoma caninum, infects dogs, feeding on blood and intestinal tissues (Roche and Layrisse, 1966). Hookworms utilise haemoglobin (Hb) as a major nutrient source, and while the mechanisms of Hb degradation in blood-feeding helminths are being elucidated (Brindley et al., 2001; Williamson et al., 2002, 2003a,b), little is known about the step immediately preceding this process, haemolysis. Haemolysins are thought to be a prerequisite for effective blood feeding in haematophagous parasites (Fetterer and Rhoads, 1997), and haemolytic activity has been identified in different parasitic phyla, including protozoans (Young and Lowrey, 1989; Rosales-Encina et al., 1992; Noronha et al., 1994; Roggwiller et al., 1998) and haematophagous insects (Gooding, 1977; de Azambuja et al., 1983; Glupov, 1996). Recently, a pore-forming lytic protein was identified from saliva of the triatomid bug, Triatoma infestans, however, the molecule is unlikely to be involved in nutrition and was not particularly effective at lysing red blood cells (Amino et al., 2002). Haemolytic activity has been investigated in extracts from two blood-feeding helminths, Schistosoma mansoni (Kasschau and Dresden, 1986), and Haemonchus contortus (Fetterer and Rhoads, 1997), but the haemolysins have not been purified or characterised in great detail. As genome and transcriptome projects proceed for parasitic helminths, gene products with haemolytic potential have been identified. mRNAs encoding small, pore-forming 0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.04.013 International Journal for Parasitology 34 (2004) 1029–1035 www.parasitology-online.com * Corresponding author. Address: Division of Infections Diseases and Immunology, Helminth Biology Laboratory, Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Qld 4006, Australia. Tel.: þ 61-7-3845-3702; fax: þ61-7-3845-3507. E-mail address: alexl@qimr.edu.au (A. Loukas).