Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity.

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Digestive proteases of blood-feeding nematodes.

Blood-feeding parasites employ a battery of proteolytic enzymes to digest the contents of their bloodmeal. Host haemoglobin is a major substrate for these proteases and, therefore, a driving force in the evolution of parasite-derived proteolytic enzymes. This review will focus on the digestive proteases of the major blood-feeding nematodes - hookworms (Ancylostoma spp. and Necator americanus) and the ruminant parasite, Haemonchus contortus - but also compares and contrasts these proteases with recent findings from schistosomes and malaria parasites. Haematophagous nematodes express proteases of different mechanistic classes in their intestines, many of which have proven or putative roles in degradation of haemoglobin and other proteins involved in nutrition. Moreover, the fine specificity of the relationships between digestive proteases and their substrate proteins provides a new molecular paradigm for understanding host-parasite co-evolution. Numerous laboratories are actively investigating these molecules as antiparasite vaccine targets.

Vaccination with irradiated Ancylostoma caninum third stage larvae induces a Th2 protective response in dogs.

While X-irradiated live parasites are not an acceptable proposition for human vaccination, they offer a ready experimental system to explore mechanisms by which immunity against hookworm infection may be induced in humans. As such, we sought to further elucidate the details of this highly protective immune response induced by the irradiated vaccine in canids, with special emphasis on the cellular aspects of the response. Vaccination with irradiated L3 induced high production of antibodies and strong PBMCs proliferation to crude L3 antigen preparation. Elevated IL-4 production was also observed in vaccinated dogs, especially in relation to IFN-gamma production (IL-4/IFN-gamma ratio). Serum from vaccinated animals inhibited penetration of L3 through canine skin in vitro by 60%. Finally, vaccinated animals had a strong antibody response to ASP-2, a promising vaccine antigen that is an excretory-secretory product of L3. These results add further support the idea that the Th2 immune response is required to generate protective immunity against hookworm larvae and that ES molecules released during this developmental stage are likely targets of this response.

Ancylostoma caninum MTP-1, an astacin-like metalloprotease secreted by infective hookworm larvae, is involved in tissue migration.

Infective larvae (L3) of nematodes secrete macromolecules that are critical to infection and establishment of the parasite in the host. The dog hookworm Ancylostoma caninum secretes an astacin-like metalloprotease, Ac-MTP-1, upon activation in vitro with host serum. Recombinant Ac-MTP-1 was expressed in the baculovirus/insect cell system as a secreted protein and was purified from culture medium by two separate methods, cation-exchange fast-performance liquid chromatography and gelatin-affinity chromatography. Recombinant MTP-1 was catalytically active and digested a range of native and denatured connective tissue substrates, including gelatin, collagen, laminin, and fibronectin. A dog was immunized with recombinant Ac-MTP-1 formulated with AS03 adjuvant, and the antiserum was used to immunolocalize the anatomic sites of expression within A. caninum L3 to secretory granules in the glandular esophagus and the channels that connect the esophagus to the L3 surface and to the cuticle. Antiserum inhibited the ability of recombinant MTP-1 to digest collagen by 85% and inhibited larval migration through tissue in vitro by 70 to 75%, in contrast to just 5 to 10% inhibition obtained with preimmunization serum. The metalloprotease inhibitors EDTA and 1,10-phenanthroline also reduced the penetration of L3 through skin in vitro by 43 to 61%. The data strongly suggest that Ac-MTP-1 is critical for the invasion process of hookworm larvae, and moreover, that antibodies against the enzyme can neutralize its function and inhibit migration.

A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms.

Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb alpha and beta chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

A secreted protein from the human hookworm necator americanus binds selectively to NK cells and induces IFN-gamma production.

Parasitic helminths induce chronic infections in their hosts although, with most human helminthiases, protective immunity gradually develops with age or exposure of the host. One exception is infection with the human hookworm, Necator americanus, where virtually no protection ensues over time. Such observations suggest these parasites have developed unique mechanisms to evade host immunity, leading us to investigate the role of the excretory/secretory (ES) products of adult N. americanus in manipulating host immune responses. Specifically, we found that a protein(s) from ES products of adult N. americanus bound selectively to mouse and human NK cells. Moreover, incubation of purified NK cells with N. americanus ES products stimulated the production of augmented (4- to 30-fold) levels of IFN-gamma. This augmentation was dependent on the presence of both IL-2 and IL-12 and was endotoxin-independent. This is the first report of a pathogen protein that binds exclusively to NK cells and the first report of a nematode-derived product that induces abundant levels of cytokines from NK cells. Such an interaction could provide a means of cross-regulating deleterious Th2 immune responses in the host, thereby contributing to the long-term survival of N. americanus.

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion.

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term "nemepsins" for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes the fecundity and growth of worms.

We expressed a catalytically active cysteine protease, Ac-CP-2, from the blood-feeding stage of the canine hookworm Ancylostoma caninum and vaccinated dogs with the purified protease. Dogs acquired high-titer, antigen-specific antibody responses, and adult hookworms recovered from the intestines of vaccinated dogs were significantly smaller than hookworms from control dogs. There was also a marked decrease in fecal egg counts and the number of female hookworms in vaccinated dogs. Ac-CP-2 is expressed by the parasite in the brush-border membrane of its alimentary canal, and anti-Ac-CP-2 antibodies were bound to the gut of hookworms from vaccinated dogs, which suggests that these antibodies were ingested by the parasites with their blood meal. IgG from vaccinated dogs decreased proteolytic activity against a peptide substrate by 73%, which implies that neutralizing antibodies were induced by vaccination. These results indicate that cysteine proteases involved in parasite nutrition are promising candidates as vaccines against hookworm disease.