Identification of a DAF-7 ortholog from the hookworm Ancylostoma caninum.

Infective hookworm L3 encounter a host specific signal during invasion that re-activates suspended developmental pathways. Response to this cue is critical for the successful infection and completion of the life cycle in the host. In the free-living nematode Caenorhabditis elegans, recovery from the developmentally arrested dauer stage in response to environmental cues is analogous to the resumption of development in invading hookworm L3. Transforming growth factor beta (TGF-beta) and insulin-like signalling pathways mediate dauer formation and recovery. An insulin-like signalling pathway mediates L3 activation in hookworms. To determine the role of TGF-beta signalling in hookworm infection, an ortholog of the C. elegans TGF-beta signalling molecule daf-7 was cloned and characterised. Sequence from a hookworm expressed sequence tag was used to design specific primers for PCR amplification of Ac-daf-7 from Ancylostoma caninum infective L3 cDNA. Amplicons from the 5' and 3' ends were cloned, sequenced, and combined to create a full-length composite Ac-daf-7 cDNA sequence. The 1,634 nucleotide cDNA encoded a 355 amino acid open reading frame with significant homology to Ce-DAF-7 and other TGF-beta signalling molecules. The deduced amino acid sequence contained seven conserved cysteines characteristic of TGF-beta family members, as well as two additional conserved cysteines found in members of the TGF-beta/activin subfamily. Ac-DAF-7 contains a characteristic C-terminal ligand domain that is predicted to be released from a propeptide by proteolytic cleavage at a tetrabasic cleavage site. Ac-daf-7 mRNA was strongly detected by reverse transcriptase PCR in L3 and serum stimulated L3 cDNA, and weakly in cDNA from L1 and adult life cycle stages. Antiserum against Escherichia coli expressed recombinant Ac-DAF-7 detected the mature protein in L3 and adult soluble extracts, but not in excretory/secretory products from serum stimulated L3 or adults. Increased expression in arrested L3 stages suggests that Ac-daf-7 is important for developmental arrest.

Cloning and characterisation of an aspartyl protease inhibitor (API-1) from Ancylostoma hookworms.

Hookworm infection persists as a public health problem in developing nations. Vaccine-based strategies offer the best chance of long-term control. Aspartyl protease inhibitors from parasitic nematodes are highly immunogenic, and have been suggested as potential vaccine antigens. An aspartyl protease inhibitor, API-1, was cloned and characterised from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. Using sequence from the hookworm expressed sequence tag project, specific primers were designed and used to amplify Ac-api-1 from A. caninum infective L3 cDNA by PCR. Amplicons from the 5' and 3' ends were cloned, sequenced, and combined to create an 874-bp full-length composite sequence of the Ac-api-1 gene. The A. ceylanicum api-1 cDNA of 878 bp was cloned from L3 cDNA using the A. caninum primers. The amino acid sequences of hookworm orthologues were nearly identical, and database searching indicated they belonged to the aspin family, a group of nematode specific aspartyl protease inhibitors that includes the Ascaris pepsin inhibitor PI-3. Ac-api-1 mRNA was detected by reverse transcriptase PCR in eggs, L1, L3 and adult life cycle stages. A polyclonal antiserum against Escherichia coli expressed recombinant Ac-API-1 detected the protein in adult A. caninum excretory/secretory products, but not in those from activated infective larvae. Immunolocalisation experiments using the antiserum indicated that Ac-API-1 is present primarily in the pseudocoelomic fluid in adult hookworms. Soluble, yeast-expressed Ac-API-1 failed to inhibit pepsin or a hookworm gut aspartyl protease in vitro, but inhibited approximately 30% of the proteolytic activity of adult excretory/secretory products. The pseudocoleomic location, presence in all life cycle stages, lack of inhibitory activity against pepsin, and inhibitory activity against excretory/secretory products suggest that Ac-API-1 inhibits an unidentified, putative aspartyl protease secreted by adult hookworms, and may be released as an enzyme-inhibitor complex. The highly immunogenic properties of nematode aspins suggest that Ac-API-1 represents a promising target for a recombinant hookworm vaccine.