The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

The development of a vaccine would provide an important new tool for the control of human hookworm infection. On the basis of successful vaccination of laboratory animals with living irradiated, third-stage hookworm larvae (L3), we examined the antibody responses of individuals from hookworm endemic areas of Brazil and China against the most abundant L3 secreted antigens, the ancylostoma secreted proteins, ASP-1 and ASP-2. Logistic regression was used to investigate the effects of antibody isotype responses to ASPs on the risk of an individual harboring heavy hookworm infection. A significant protective association was observed between increasing anti-ASP-2 IgE levels and the risk of heavy hookworm infection. To confirm that ASP-2 is a protective antigen, laboratory dogs were immunized with recombinant ASP-2 formulated with the GlaxoSmithKline Adjuvant, AS03. Sera obtained from the immunized dogs exhibited high geometric mean antibody titers, immunoprecipitated native ASP-2 from L3 extracts and localized the site of ASP-2 expression to the glandular esophagus and body channels exiting to the cuticle. The sera also exhibited an increased ability to inhibit migration of L3 through tissue in vitro relative to sera from AS03-injected controls. Upon L3 challenge, the ASP-2 vaccinated dogs exhibited significant reductions in fecal egg counts and intestinal hookworm burden. These findings provide strong support for the development of an effective recombinant vaccine against hookworm infection in humans.

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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.

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 ␣ and ␤ 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.

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 ...

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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.