Hookworm aspartic proteases & their contribution to host specificity

Williamson, Angela Louise (2002). Hookworm aspartic proteases & their contribution to host specificity PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author Williamson, Angela Louise
Thesis Title Hookworm aspartic proteases & their contribution to host specificity
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Dr Peter O'Donoghue
Dr Alex Loukas
Total pages 145
Collection year 2002
Language eng
Subjects L
300508 Parasitology
770704 Control of pests and exotic species
Formatted abstract

Hookworms are an important group of intestinal parasites and, like many other pathogens, are highly host-specific. Necator americanus only produces patent infections in humans under natural conditions; while Ancylostoma caninum only reaches patency in canids. However, A. caninum infective larvae (L3) readily penetrate human skin, and although some parasites reach the gut, they are never sexually mature. Likewise, dogs can be experimentally infected with A'; americanus L3, however, the vast majority do not mature to fertility. It was therefore hypothesized that the development of L3 to the blood-feeding adult stage may partly depend on host-specific nutritional requirements. Other blood-feeding parasites including schistosomes and Plasmodium digest host haemoglobin (Hb) using aspartic proteases. Hookworms might also utilize similar proteolytic mechanisms to cleave host Hb, and furthermore, the complementarity between hookworm haemoglobinases and their specific host Hb molecules may be a contributing factor governing which host species a hookworm can develop and mature in.      


cDNAs encoding cathepsin D-like aspartic proteases from A. caninum (Ac-apr-1) and N. americanus (Na-apr-1 and Na-apr-2) were expressed as secreted pro-peptidases in baculovirus and were active against a cathepsin D fluorescent substrate (Abz-Ile-Glu- Phe-nPhe-Arg-Leu-NH2). The abilities of the recombinant enzymes to cleave Hb were assessed by incubating each purified protease with Hb from both permissive and non permissive hosts. The two orthologous cathepsin D proteases from A. caninum (Ac-APR- 1) and N. americanus (Na-APR-1) exhibited 80% identity over the entire protein but were 100% identical in their active site residues. Both recombinant zymogens autoactivated at pH 3.6 to form proteases that cleaved Hb at a pH optimum of 5.5. Ac- APR-1 digested canine Hb almost twice as efficiently as human Hb. In contrast, Na- APR-1 cleaved human Hb almost twice as efficiently as dog Hb. Furthermore, both proteases were shown to degrade collagens, albumin, and fibrinogen. Both enzymes equally degraded human fibrinogen and albumin, whereas Na-APR-1 was less efficient at degrading dog fibrinogen and albumin than Ac-APR-1. Na-APR-1 degraded human collagen more efficiently than Ac-APR-1. Specific cleavage sites were mapped to the ∞- and β-chains of human and dog Hb. Despite some conserved cleavage sites, many cleavage events were unique to just one protease. Ac- and Na-APR-1 accommodated substrates with serine or alanine in the P3 position of ∞-Hb, but Na-APR-1 was unique in  its affinity for histidine at P3. Two cleavage sites (LDKF*SLASV and LDKF*FAAV) on human and canine Hb ∞-chains respectively, were synthesized to kinetically confirm the differences in substrate specificity between the two proteases. Ac-APR-1 cleaved the dog Hb peptide 6-fold more efficiently than Na-APR-1. Na-APR-1 cleaved the human Hb peptide 4-fold more efficiently than Ac-APR-1. Antisera were raised to the recombinant proteases and purified IgG completely inhibited the enzymatic activities against the fluorogenic substrate but did not inhibit their activity against Hb. Antisera partially inhibited (20-30%) L3 migration through hamster skin in vitro. Both Ac-APR-1 and Na-APR-1 primarily localized to the gut and amphidial glands in adult worms in immunohistochemical localization, supporting their roles as haemoglobinases.     


 A pepsinogen-like aspartic protease from N americanus (Na-apr-2) was also cloned, expressed, and its activity characterized. The predicted Na-APR-2 protein had 30% identity to Na-APR-1 and 45% to pepsinogen from Haemonchus contortus. Recombinant Na-APR-2 autoactivated at pH 3.6 to form a protease that digested human Hb almost twice as efficiently as dog Hb. Differences in the pH profiles and Hb cleavage sites of Na-APR-2 (compared to Na-APR-1) were also found, suggesting an ordered pathway for hookworm gut Hb degradation. Na-APR-2 is also expressed in infective larvae and it degraded collagen, albumin, and fibrinogen. Antiserum was raised against recombinant, purified Na-APR-2. Purified anti-Na-APR-2 IgG completely inhibited the enzymatic activity against Abz-Ile-Glu-Phe-nPhe-Arg-Leu-NH2 but did not inhibit its activity against Hb. Anti-Na-APR-2 IgG also partially inhibited the enzymatic activity of Na-APR-1 and Ac-APR-1 against the fluorogenic substrate. Anti-Na-APR-1 and anti-Ac-APR-1 IgG inhibited the enzymatic activity of Na-APR-2 against the fluorogenic substrate by 40%. Anti-Na-APR-2 inhibited migration of N. americanus L3 through hamster skin by 50%, and A. caninum L3 migration by 30%. Na-APR-2 localized primarily to the gut and amphidial glands in male and female adult worms, supporting a role as a haemoglobinase in vivo.      


A likely mechanism that may contribute to the host specificity of hookworms, and perhaps many other pathogens, has now been identified. In this case, complementarity between Hb molecules and the parasite proteases which cleave them appears to influence which hosts a given hookworm species can survive in. Incompatibility between proteases and target host molecules might also explain the limited host range of other blood feeding pathogens. 

Keyword Hookworms
Additional Notes Missing page 57 in the original thesis.

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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Created: Fri, 24 Aug 2007, 17:53:13 EST