Astacin metalloproteases in human-parasitic nematodes

Abstract

Parasitic nematodes infect over 2 billion individuals worldwide, primarily in low-resource areas, and are responsible for several chronic and potentially deadly diseases. Throughout their life cycle, these parasites are thought to use astacin metalloproteases, a subfamily of zinc-containing metalloendopeptidases, for processes such as skin penetration, molting, and tissue migration. Here, we review the known functions of astacins in human-infective, soil-transmitted parasitic nematodes - including the hookworms Necator americanus and Ancylostoma duodenale, the threadworm Strongyloides stercoralis, the giant roundworm Ascaris lumbricoides, and the whipworm Trichuris trichiura - as well as the human-infective, vector-borne filarial nematodes Wuchereria bancrofti, Onchocerca volvulus, and Brugia malayi. We also review astacin function in parasitic nematodes that infect other mammalian hosts and discuss the potential of astacins as anthelmintic drug targets. Finally, we highlight the molecular and genetic tools that are now available for further exploration of astacin function and discuss how a better understanding of astacin function in human-parasitic nematodes could lead to new avenues for nematode control and drug therapies.

Keywords: Astacins; Filarial nematodes; Helminths; Hookworms; Human-parasitic nematodes; Molting; Neglected tropical diseases; Skin penetration; Strongyloides; Zinc metalloproteases.

Figure 1.. The life cycles of selected human-parasitic nematodes, with potential roles of astacins indicated.

Schematics of the life cycles of the skin-penetrating threadworm Strongyloides stercoralis (A), a skin-penetrating hookworm (B), the passively ingested worm Ascaris lumbricoides (C), and a vector-borne filarial worm (D). L1-L4 = 1^st-4^th larval stages; iL3 = infective third larval stage. Life cycle stages that exist inside the host are indicated by the person; life cycle stages that exist in the environment are indicated by the grass and soil; life cycle stages that exist in an insect vector are indicated by the mosquito. The stages of the life cycle where astacins have been demonstrated or hypothesized to function are indicated by the colored symbols, as defined in the key. Life cycle schematics are adapted with permission from Wheeler et al., 2022 (Wheeler et al., 2022) and were generated in BioRender.

Figure 2.. The structure of astacin proteins.

A. Schematic of the functional domains found in three astacin proteins: A. astacus astacin, S. stercoralis strongylastacin, and C. elegans HCH-1. All astacins contain an N-terminal signal peptide (S), a prodomain (PD), and a zinc-dependent catalytic peptidase domain (CPD). Many astacins also contain additional domains, including epidermal growth factor-like (EGF); complement C1r/C1s, Uegf, Bmp1 (CUB); and threonine-rich (TRD) domains. Domains are shown ordered in the 5' to 3' direction; domain sizes are approximate and inter-domain intervals are not to scale. Figure is adapted with permission from Gomez Gallego et al., 2005 (Gomez Gallego et al., 2005). B. Protein alignment of S. stercoralis strongylastacin and A. astacus astacin. Alignment was generated using UniProt. Domains were identified using Prosite and InterPro; motifs were identified based on motif descriptions in Gomis-Rüth and Stöcker, 2023 (Gomis-Rüth and Stöcker, 2023). C. Structure of the predicted peptidase domain of S. stercoralis strongylastacin. The His and Glu residues that bind to the zinc ion are in blue; selected residues of the methionine turn are in magenta. The N- and C-terminal regions of astacin are depicted by yellow arrows. Figure is reprinted with permission from Gomez Gallego et al., 2005 (Gomez Gallego et al., 2005).

Figure 3.. Expansion of the astacin family in skin-penetrating nematodes.

Phylogeny of astacin proteins in the phyla Nematoda (including free-living and parasitic nematodes in Clades I–V) and Platyhelminthes (including cestodes and trematodes). Parasitic nematodes in Clade IV (which includes Strongyloides species) and Clade V (which includes hookworms) have greatly expanded astacin families. Selected astacins that have been functionally studied are labeled. S. stercoralis strongylastacin is thought to contribute to skin penetration (Gomez Gallego et al., 2005; McClure et al., 2023; McKerrow et al., 1990), A. caninum MTP-1 is thought to contribute to skin penetration and tissue migration (Hawdon et al., 1995; McClure et al., 2023; Williamson et al., 2006; Zhan et al., 2002), B. malayi NAS-36 contributes to molting (Stepek et al., 2011), and O. volvulus AST-1 is thought to contribute to tissue migration (Borchert et al., 2007). Figure is reprinted with permission from Martín-Galiano and Sotillo, 2022 (Martin-Galiano and Sotillo, 2022).

Figure 4.. RNAi-mediated knockdown of the H. contortus gene nas-33 causes molting defects.

A. Knockdown of nas-33 in H. contortus first-stage larvae in an RNAi feeding assay resulted in a molting defect in which the old cuticle remained partly attached to the second-stage larva after the molt (Huang et al., 2021). B-D. Enlarged images of the indicated regions in A; sites where the old cuticle remained attached to the second-stage larva are indicated with white arrows (Huang et al., 2021). Images are reprinted from Huang et al., 2021 (Huang et al., 2021).

Figure 5.. Metalloprotease inhibitors prevent skin penetration by S. stercoralis iL3s.

A. Incubation of ES products from S. stercoralis iL3s with metalloprotease inhibitors blocks their proteolytic activity, suggesting that protein degradation by iL3s requires metalloproteases. ES products isolated from S. stercoralis iL3s were incubated with either metalloprotease inhibitors (2.5 mM 1,10-phenanthroline; 10 mM HONHCOCH (CH₂CH₂CH Me₂) CO-Ala-Gly-NH₂; or 1 mM HO₂C-CH₂-Phe-Leu), serine protease inhibitors (5 mM PMSF, 100 μg/mL Aprotinin, or 100 μg/mL α-1-proteinase inhibitor), a cysteine protease inhibitor (5 mM NEM), or an aspartic protease inhibitor (50 μg/mL pepstatin). The ES products were then exposed to the substrate azocoll, and the % inhibition of azocoll degradation was determined. Only exposure to metalloprotease inhibitors blocked azocoll degradation. B. Incubation of S. stercoralis iL3s with metalloprotease inhibitors blocks skin penetration in an ex vivo skin penetration assay. iL3s incubated with either metalloprotease inhibitors (2 mM 1,10-phenanthroline; 2 mM HONHCOCH (CH₂CH₂CH Me₂) CO-Ala-Gly-NH₂; or 2 mM HO₂C-CH₂-Phe-Leu) or a serine protease inhibitor (2 mM PMSF) were placed on rat skin, and the % inhibition of skin penetration was determined based on the number of iL3s that penetrated the skin. Only exposure to the metalloprotease inhibitors blocked skin penetration. For A-B, percent inhibition was calculated for each chemical relative to the vehicle control. Data are replotted with permission from McKerrow et al., 1990 (McKerrow et al., 1990).