The hookworm Ancylostoma ceylanicum intestinal transcriptome provides a platform for selecting drug and vaccine candidates

Abstract

Background: The intestine of hookworms contains enzymes and proteins involved in the blood-feeding process of the parasite and is therefore a promising source of possible vaccine antigens. One such antigen, the hemoglobin-digesting intestinal aspartic protease known as Na-APR-1 from the human hookworm Necator americanus, is currently a lead candidate antigen in clinical trials, as is Na-GST-1 a heme-detoxifying glutathione S-transferase.

Methods: In order to discover additional hookworm vaccine antigens, messenger RNA was obtained from the intestine of male hookworms, Ancylostoma ceylanicum, maintained in hamsters. RNA-seq was performed using Illumina high-throughput sequencing technology. The genes expressed in the hookworm intestine were compared with those expressed in the whole worm and those genes overexpressed in the parasite intestine transcriptome were further analyzed.

Results: Among the lead transcripts identified were genes encoding for proteolytic enzymes including an A. ceylanicum APR-1, but the most common proteases were cysteine-, serine-, and metallo-proteases. Also in abundance were specific transporters of key breakdown metabolites, including amino acids, glucose, lipids, ions and water; detoxifying and heme-binding glutathione S-transferases; a family of cysteine-rich/antigen 5/pathogenesis-related 1 proteins (CAP) previously found in high abundance in parasitic nematodes; C-type lectins; and heat shock proteins. These candidates will be ranked for downstream antigen target selection based on key criteria including abundance, uniqueness in the parasite versus the vertebrate host, as well as solubility and yield of expression.

Conclusion: The intestinal transcriptome of A. ceylanicum provides useful information for the identification of proteins involved in the blood-feeding process, representing a first step towards a reverse vaccinology approach to a human hookworm vaccine.

Keywords: Ancylostoma ceylanicum; Hookworm; Intestine; Transcriptome; Vaccine candidate.

Fig. 1

The intestines were isolated from A. ceylanicum male worms avoiding esophagus and reproductive organs (a), and total RNA was extracted and treated with DNase. The integrity of the total intestinal RNA was analyzed by electrophoresis and the RNA Integrity Number (RIN) for the purified RNA was 8.6 (b)

Fig. 2

Gene ontologies of those genes expressed predominantly in the intestine versus the whole adult worm, expressed as FPKM values

Fig. 3

Expression levels of various peptidase classes in the intestine of adult A. ceylanicum compared to those in the whole worm

Fig. 4

TrSPP transporter categories: FPKM relative proportions for genes significantly expressed in the intestine of adult A. ceylanicum compared to the whole worm

Fig. 5

Phylogenetic tree comparing Na-GST-1 with 13 newly identified GSTs expressed in the A. ceylanicum intestine, with branch support values in red. ANCCEY_00737 shares the highest similarity with Na-GST-1 (66 % amino acid identity)

Fig. 6

Phylogenetic tree comparing the eleven most abundant ASPs expressed in the adult A. ceylanicum intestine with ASPs previously identified in A. caninum adults (Ac-ASP-3, -4, -5 and -6) and larvae (Ac-ASP-1 and Ac-ASP-2), with branch support values in red