Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Abstract

Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met 26 in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.Hookworm infection remains a major global health problem, and over one billion people are reportedly infected in developing countries (9, 14). Hookworms, which are bloodfeeding intestinal nematodes, are a major cause of iron deficiency anemia and malnutrition (15,20,(59)(60)(61)64). While the anemia is presumably due to the cumulative effect of chronic intestinal blood loss, the molecular mechanisms underlying the pathogenesis of hookworm malnutrition remain unknown. Although it has been suggested that hookworm malnutrition and growth delay occur secondary to chronic iron deficiency, particularly in children, evidence from prior clinical studies suggests that hookworm infection is also associated with various degrees of intestinal malabsorption (18,35,54,57,62). It has been hypothesized that this hookworm malabsorption syndrome might occur secondary to mucosal inflammation triggered by the adult worm attached to the intestinal epithelium or might be a result of secretion of parasite inhibitors of host digestive enzymes (18).As part of a series of ongoing studies aimed at characterizing adult hookworm secretory proteins, a cDNA corresponding to the gene encoding a putative Kunitz-type serine protease inhibitor was previously identified from adult Ancylostoma ceylanicum RNA by using a PCR-based approach (48). The A. ceylanicum Kunitz-type inhibitor (AceKI) cDNA was expressed in Escherichia coli, and the recombinant protein was found to inhibit the pancreatic enzymes chymotrypsin, pancreatic elastase, and trypsin in vitro, with equilibrium inhibitory dissociation constant (K i ) values ranging from picomolar levels to low nanomolar levels. The native AceKI prote...

The kunitz domain protein BLI-5 plays a functionally conserved role in cuticle formation in a diverse range of nematodes

“…ceylanicum [18,34], these kunitz-type inhibitor domains have been predicted to have an essential role in nematode development, a role also envisaged for this domain in the BLI-5 orthologues described in this study.…”

Purification and Molecular Cloning of and Immunization with Ancylostoma ceylanicum Excretory-Secretory Protein 2, an Immunoreactive Protein Produced by Adult Hookworms

“…For example, if AceES-2 is a virulence factor that has a role in bloodfeeding, neutralization of its function might be expected to have a greater effect on anemia than on weight loss (assuming that the worms continue to survive with reduced feeding efficiency). Conversely, it has recently been demonstrated that vaccination with the Kunitz inhibitor AceKI leads to improved weight gain but has little effect on anemia (22), which is consistent with the demonstrated activity of this protein against host digestive enzymes (47). Together, these results suggest that the pathogenesis of hookworm anemia and growth delay are due to distinct molecular mechanisms and that vaccination against specific virulence factors may allow these disease parameters to be experimentally decoupled.…”

Host Cytokine Production, Lymphoproliferation, and Antibody Responses during the Course of Ancylostoma ceylanicum Infection in the Golden Syrian Hamster

“…As the human hookworm A. duodenale is both orally infective as well as infective through the percutaneous route, this approximates a natural route of infection. More importantly, this model reproduces weight and blood loss that result from human infection with A. duodenale.In addition to modeling the host-parasite relationship for human hookworm infection, the hamsters have been used with some success for investigating protective immunity from a new generation of recombinant anti hookworm vaccines (2,8,9,13,22,33). However, detailed immunological investigations have not been conducted in this model due of the lack of available immunological reagents.…”