Ancylostoma caninum MTP-1, an Astacin-Like Metalloprotease Secreted by Infective Hookworm Larvae, Is Involved in Tissue Migration

Abstract

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.

Necator americanus: The Na-ASP-2 protein secreted by the infective larvae induces neutrophil recruitment in vivo and in vitro

“…In particular, tissue-invading hookworm L3 release metalloproteases and ASPs. The function of the A. caninum metalloprotease-1, Ac-MTP-1, has recently been characterized by us (Williamson et al, 2006). We have shown that MTP-1 favors tissue penetration: anti-MTP-1 antiserum inhibited the ability of the recombinant protein to digest collagen by 85% and inhibited larval migration through tissue in vitro by 75% (Williamson et al, 2006).…”

“…The function of the A. caninum metalloprotease-1, Ac-MTP-1, has recently been characterized by us (Williamson et al, 2006). We have shown that MTP-1 favors tissue penetration: anti-MTP-1 antiserum inhibited the ability of the recombinant protein to digest collagen by 85% and inhibited larval migration through tissue in vitro by 75% (Williamson et al, 2006). Similarly, rat and dog antiserum against Na-ASP-2 was able to inhibit larval migration through tissue in an in vitro system Goud et al, 2005).…”

Serine Protease-mediated Host Invasion by the Parasitic Nematode Steinernema carpocapsae

“…Nematodes in the arrested stage do not express sc-sp-1; however, this gene was overexpressed in recovered nematodes, particularly those in the gut lumen preparing to invade the insect hemocoelium. In other parasitic nematodes, the role for serine proteases in tissue invasion was inferred based on expression analysis (52)(53)(54). In vitro assays showed that, although the resistant stage does not express scsp-1, the nematodes initiate expression shortly after stimulation with insect tissues.…”

The important role of matrix metalloproteinases in nematode parasites

“…Many of these parasites to complete their life cycle have to migrate or invade through various organs of their hosts. During this journey, expression and release of MMPs by various parasitic nematodes have been associated with the pathology resulting from histolysis (Williamson et al, 2006). Genetic analysis and cDNA cloning of MMPs have been evaluated in few parasitic nematodes (Salter et al, 2002;Uparanukraw et al, 2001;Wang et al, 2001).…”