A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy

Abstract

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST—of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol—and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion.

Introduction

Hookworms are blood-feeding nematodes which inhabit the small intestines of their definitive mammalian hosts. Infective L3 penetrate the host's skin and migrate via the circulatory system to reside in the duodenum as adult stage worms (1–1.5 cm in length). Adult parasites bury their anterior ends beneath the mucosa of the bowel, rupture capillaries and feed on the extravasated blood. The pathogenesis of hookworm infection is a direct consequence of the blood loss which occurs during attachment and feeding. In developing countries, hookworms are the leading cause of iron deficiency anaemia, which, in heavy infections, can cause developmental and mental retardation in children as well as adverse maternal–foetal outcomes in pregnant women (Hotez et al., 2004).

Current control strategies for hookworm are limited mainly to the treatment of infected patients with anthelmintic drugs. However, due to increasing drug resistance in parasitic nematodes of livestock and the perception that this may occur in helminths of humans, as well as the absence of naturally acquired immunity in most exposed people (Loukas et al., 2005b), the major focus of research has shifted towards developing an effective hookworm vaccine. Through the auspices of the Human Hookworm Vaccine Initiative, an antigen (Na-ASP-2) derived from the L3 of Necator americanus, a major hookworm species of humans, was selected for progression to clinical trials (Bethony et al., 2005, Goud et al., 2005). Na-ASP-2 is expressed exclusively by the L3 and is only partially efficacious at reducing worm burdens in vaccinated animals. Therefore, a useful human hookworm vaccine will likely require a second antigen, preferably one derived from the adult blood-feeding stage of the parasite (Hotez et al., 2003b, Loukas et al., 2005b). Hookworms ingest red blood cells, lyse the cells in their intestines via pore-forming proteins (Don et al., 2004) and digest the liberated haemoglobin using a semi-ordered cascade of proteases, some of which have been characterised in vitro (Williamson et al., 2004). Vaccine trials in dogs with some of the recombinant haemoglobin-degrading proteases (haemoglobinases) have shown encouraging levels of efficacy (Loukas et al., 2004, Loukas et al., 2005a). Moreover, a related nematode that parasitizes livestock, Haemonchus contortus, can be successfully vaccinated against using extracts enriched for haemoglobinases (Knox and Smith, 2001, Knox et al., 2005), and a recombinant vaccine against cattle tick targets a gut membrane glycoprotein (de la Fuente et al., 1999), lending support to the targeting of gut proteins for the development of vaccines against blood-feeding parasites.

Expressed sequence tags (ESTs) have been characterised from hookworms (Daub et al., 2000, Mitreva et al., 2005), but the majority of these sequences are derived from the L3 stage for Ancylostoma sp. (approximately 20,000—www.nematode.net). Less than 5000 ESTs from N. americanus have been described (see www.nematodes.org) and are deposited in dbEST (www.ncbi.nlm.gov/dbEST) (Parkinson et al., 2004). With a view to identifying mRNAs encoding potential new vaccine antigens from the hookworm gut, we characterised gut-specific transcripts of the two major hookworms of humans and canines, N. americanus and Ancylostoma caninum, respectively. Adult hookworms are small, which makes it very difficult to accurately dissect individual tissues, unlike the situation for larger nematodes such as H. contortus (Jasmer et al., 2001). However, with the development of laser microdissection microscopy (LMM), a technique which allows dissection of tissues and even individual cells (Jones et al., 2004), it is now possible to dissect defined organs and cells from small parasites for subsequent isolation of tissue-specific proteins and nucleic acids. The potential of this application for extracting individual cells/tissues from histological sections of helminths has been proposed (Jones et al., 2004). Here, we describe the first application of LMM to the dissection of gut tissue from adult N. americanus and A. caninum, extraction of RNA for production of cDNA libraries and comparative analyses of ESTs from each library.