Parasites and Vectors
Article number 14
Hookworms infect nearly 700 million people, causing anemia and developmental stunting in heavy infections. Little is known about the genomic structure or gene regulation in hookworms, although recent publication of draft genome assemblies has allowed the first investigations of these topics to be undertaken. The transcription factor DAF-16 mediates multiple developmental pathways in the free living nematode Caenorhabditis elegans, and is involved in the recovery from the developmentally arrested L3 in hookworms. Identification of downstream targets of DAF-16 will provide a better understanding of the molecular mechanism of hookworm infection.
Genomic Fragment 2.23 containing a DAF-16 binding element (DBE) was used to identify overlapping complementary expressed sequence tags (ESTs). These sequences were used to search a draft assembly of the Ancylostoma caninum genome, and identified two neighboring genes, snr-3 and lpp-1, in a tail-to-tail orientation. Expression patterns of both genes during parasitic development were determined by qRT-PCR. DAF-16 dependent cis-regulatory activity of fragment 2.23 was investigated using an in vitro reporter system.
The snr-3 gene spans approximately 5.6 kb in the genome and contains 3 exons and 2 introns, and contains the DBE in its 3′ untranslated region. Downstream from snr-3 in a tail-to-tail arrangement is the gene lpp-1. The lpp-1 gene spans more than 6 kb and contains 10 exons and 9 introns. The A. caninum genome contains 2 apparent splice variants, but there are 7 splice variants in the A. ceylanicum genome. While the gene order is similar, the gene structures of the hookworm genes differ from their C. elegans orthologs. Both genes show peak expression in the late L4 stage. Using a cell culture based expression system, fragment 2.23 was found to have both DAF-16-dependent promoter and enhancer activity that required an intact DBE.
Two putative DAF-16 targets were identified by genome wide screening for DAF-16 binding elements. Aca-snr-3 encodes a core small nuclear ribonucleoprotein, and Aca-lpp-1 encodes a lipid phosphate phosphohydrolase. Expression of both genes peaked at the late L4 stage, suggesting a role in L4 development. The 3′-terminal genomic fragment of the snr-3 gene displayed Ac-DAF-16-dependent cis-regulatory activity.
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Gao, X., Goggin, K., Dowling, C., Qian, J., Hawdon, J.M. (2015). Two potential hookworm DAF-16 target genes, SNR-3 and LPP-1: gene structure, expression profile, and implications of a cis-regulatory element in the regulation of gene expression. Parasites and Vectors, 8:14.
Representative Ancylostoma ceylanicum stages collected at 72 h post infection. A. Parasitic L3 stage. Note the lack of significant morphological remodeling in the anterior. B. Late parasitic L3 stage. Note the provisional buccal capsule longitudinally bisected by the cuticle lining of the former buccal cavity (arrow). C. Parasitic L4 stage. Note the complete buccal capsule characteristic of this stage (arrow).
Additional file 2.tif (91 kB)
Map of the Aca-snr-3 gene showing the location of Fragment 2.23 and the Daf-16 binding element. Exons are indicated by boxes, with coding regions in green and untranslated regions in aqua. Introns are indicated as lines. The red bar shows the approximate location of Fragment 2.23, and the inset shows its DNA sequence. The 3′ sequence of intron 2 is in italicized lowercase text, exon 3 sequence is in blue uppercase text, and the DBE in red text. The 3′ splice sequence of intron 2 is underlined, the polyadenylation signal sequence is in pink text, and the site of polyA tail addition is boxed. Numbering is from the original contig sequence.
Additiona file 3.tif (72 kB)
Predicted isoforms of Ace-LPP-1. Isoforms sequences, indicated by accession number, were obtained from Genbank and mapped to A. ceylanicum draft genomic contigs to determine gene structure. Similar exons are shown in the same color. The number of amino acids is shown above each exon. The intron length in base pairs is indicated below each intron. The location of the primers used for qPCR is indicated by arrowheads.