Molecular cloning and characterisation of in vitro immune response against astacin-like metalloprotease Ace-MTP-2 from Ancylostoma ceylanicum

Abstract

Ancylostoma ceylanicum belongs to the group of parasites commonly known as hookworms, blood-sucking nematodes which infect around 576 million people and hundreds of millions of animals. The interactions between these parasites and host immune systems are complicated and yet to be determined. Hookworm infections are usually long lasting and recurrent, due in part to their ability to synthesize macromolecules capable of modulating the host immune response. The interaction of parasite proteins with host immune systems has been proven, but so far there is no data describing the influence of astacin-like metalloproteases (expressed among different parasitic nematodes) on the human immune system. The cDNA encoding A. ceylanicum metalloprotease 2 (Ace-mtp-2) was cloned using RACE-PCR. Computational analysis was used to examine the immunogenicity and recombinant Ace-MTP-2 was used to investigate its influence on human THP-1 monocytes and macrophages. The Ace-mtp-2 gene encodes an astascin-like metalloprotease, with a theoretical molecular mass of 26.7 kDa. The protease has a putative signal peptide, 11 potential phosphorylation sites, and two disulfide bridges revealed by computational analysis. Maximal expression of Ace-mtp-2 by A. ceylanicum occurs in the adult stage of the parasite, and Western blot indicates the secretory nature of the protease. This suggests the protease is working at the host-parasite interface and would likely be exposed to the hosts immune response. Recombinant protein were expressed in Escherichia coli and Pichia pastoris. Recombinant Ace-MTP-2 amplified the in vitro release of TNFα and induced release of IFNγ by lipopolysaccharide activated THP-1 macrophages. The presence of Ace-MTP-2 in secretory products of the adult parasite and the induction of IFNγ release may suggest an important role for Ace-MTP-2 in host-parasite interactions since IFNγ is suggested to be responsible for the protective immune response against adult hookworms.