A family of cathepsin B cysteine proteases expressed in the gut of the human hookworm, Necator americanus

mRNAs encoding cathepsin B-like cysteine proteases (CatBs) are abundantly expressed in the genomes of blood-feeding nematodes. Recombinant CatBs have been partially efficacious in vaccine trials in animal models of hookworm infection, supporting further investigation of these enzymes as new control tools. We recently described a family of four distinct CatBs (Na-CP-2, -3, -4,-5) from the human hookworm, Necator americanus. Here we show that these N. americanus CatBs form a robust clade with other hookworm CatBs and are most similar to intestinal CatBs from Haemonchus contortus. All four mRNAs (Na-cp-2, -3, -4 and -5) are up-regulated during the transition from a free-living larva to a blood-feeding adult worm and are also expressed in gut tissue of adult N. americanus that was dissected using laser microdissection microscopy. Recombinant Na-CP-3 was expressed in soluble, secreted form in the yeast Pichia pastoris, while Na-CP-2, -4 and -5 were expressed in insoluble inclusion bodies in Escherichia coli. Recombinant Na-CP-3 was not catalytically active when secreted by yeast but underwent auto-activation to an active enzyme at low pH in the presence of dextran sulphate. Activated Na-CP-3 digested gelatin and cleaved the fluorogenic substrate Z-Phe-Arg-aminomethylcoumarin (AMC) but not Z-Arg-Arg-AMC. Recombinant Na-CP-3 did not digest intact hemoglobin but digested globin fragments generated by prior hydrolysis with N. americanus aspartic hemoglobinases. Antibodies raised in mice to all four recombinant proteins showed minimal cross-reactivity with each other, and each antiserum bound to the intestine of adult N. americanus, supporting the intestinal expression of their mRNAs. These data show that N. americanus expresses a family of intestinal CatBs, many of which are likely to be involved in nutrient acquisition and therefore are potential targets for chemotherapies and vaccines. (C) 2008 Elsevier B.V. All rights reserved.