Biochemical Characterization and Vaccine Potential of a Heme-Binding Glutathione Transferase from the Adult Hookworm Ancylostoma caninum

Abstract

We report the cloning and expression of Ac-GST-1, a novel glutathione S-transferase from the adult hookworm Ancylostoma caninum, and its possible role in parasite blood feeding and as a vaccine target. The predicted Ac-GST-1 open reading frame contains 207 amino acids (mass, 24 kDa) and exhibited up to 65% amino acid identity with other nematode GSTs. mRNA encoding Ac-GST-1 was detected in adults, eggs, and larval stages, but the protein was detected only in adult hookworm somatic extracts and excretory/secretory products. Using antiserum to the recombinant protein, Ac-GST-1 was immunolocalized to the parasite hypodermis and muscle tissue and weakly to the intestine. Recombinant Ac-GST-1 was enzymatically active, as determined by conjugation of glutathione to a model substrate, and exhibited a novel high-affinity binding site for hematin. The possible role of Ac-GST-1 in parasite heme detoxification during hemoglobin digestion or heme uptake prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs with Ac-GST-1 resulted in a 39.4% reduction in the mean worm burden and 32.3% reduction in egg counts compared to control dogs following larval challenge, although the reductions were not statistically significant. However, hamsters vaccinated with Ac-GST-1 exhibited statistically significant worm reduction (53.7%) following challenge with heterologous Necator americanus larvae. These studies suggest that Ac-GST-1 is a possible drug and vaccine target for hookworm infection.Hookworm infection is a major cause of disease burden for animals and humans. An estimated 740 million cases of human hookworm infection occur worldwide (12). Most of the pathology attributed to hookworm infection results from intestinal blood loss caused by the adult stages of the parasite (21, 32). The adult hookworm is specially adapted to ingest red blood cells and feed on the intracellular contents and has evolved to produce a battery of molecules for this purpose (22,42). For instance, the parasite uses its buccal capsule to attach to the intestinal mucosa and submucosa, where it mechanically ruptures capillaries and arterioles. From unique cephalic glands, the adult hookworm releases anticoagulants and anti-platelet-aggregating agents into the attachment site (10, 34). The parasite subsequently ruptures red blood cells through the action of a unique hemolysin (13) and then degrades the released hemoglobin through a carefully orchestrated cascade of hemoglobinases (43). This sequence of events is central to the pathogenesis of hookworm disease, which results almost entirely from hookworm-induced blood loss leading to iron deficiency anemia (35).The trichostrongyle Hemonchus contortus is a major cause of anemia and weight loss in small ruminants. Like hookworms, H. contortus produces numerous mechanistically distinct proteases that are thought to digest hemoglobin (27). Recently, adult H. contortus was shown to produce a novel glutathione S-transferase (Hc-GST-1), which has a high-affinity binding site for hematin ...

Molecular Cloning, Biochemical Characterization, and Partial Protective Immunity of the Heme-Binding GlutathioneS-Transferases from the Human HookwormNecator americanus

“…Pichia-derived recombinant Na-GST-1, Na-GST-2, and Na-GST-3 were tested in two separate trials for the protection they afforded against N. americanus L3 challenge in an adapted hamster model established at the Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (IPD-CCDCP) (66), under an approved protocol as described previously (65,69). Because of limited capacity in the animal facility, which could not hold more than 60 hamsters at a time, Na-GST-1 and Na-GST-2/-3 were tested separately against an adjuvant-only negative control under the same conditions in each trial.…”

“…Our previous studies have identified a nematode-specific class of parasitic GSTs exhibiting a high affinity for heme (59,69). X-ray structural data suggest that heme binding is due to the association of tetrapyrrole compounds with a large hydrophobic binding pocket that forms during homodimerization of two hookworm GST subunits (3).…”

“…Heme is released during the breakdown of host hemoglobin. Since heme is a potent enzyme inhibitor and generator of toxic ROS (44), it is possible that blood-feeding parasitic nematodes, like hookworms, employ GST proteins in heme detoxification (69). In contrast, insoluble cofactors like heme must also be transported by lipophilic proteins, such as GSTs, in order to be incorporated in essential enzymes, such as cytochrome c, peroxidases, and the numerous globins known to be present in nematode proteomes (45).…”

“…A GST expressed from the canine hookworm Ancylostoma caninum was shown to exhibit a strong affinity for heme, and vaccination with a recombinant form of this GST, Ac-GST-1, protected both dogs against A. caninum larval challenge infection (39% worm burden reduction) and hamsters against N. americanus larval challenge infection (51 to 54% worm burden reduction) (65,69). Here, we report the cloning and recombinant expression of three GSTs, Na-GST-1, Na-GST-2, and Na-GST-3, from the human hookworm N. americanus, based on their sequence similarities to Ac-GST-1.…”

Expression, purification, and characterization of theNecator americanusaspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine

“…11 These findings led to the development of an experimental human hookworm vaccine comprised of Na-APR-1, together with N. americanus glutathione S-transferase 1 (Na-GST-1) a unique hookworm enzyme that functions in heme binding and detoxification. [12][13][14][15] The goal of the vaccine strategy is to elicit neutralizing antibodies to both enzymes which would inhibit the parasites ability to detoxify and eliminate heme as well as utilize host hemoglobin for survival.…”