Identification of hookworm DAF-16/FOXO response elements and direct gene targets

Abstract

Background: The infective stage of the parasitic nematode hookworm is developmentally arrested in the environment and needs to infect a specific host to complete its life cycle. The canine hookworm (Ancylostoma caninum) is an excellent model for investigating human hookworm infections. The transcription factor of A. caninum, Ac-DAF-16, which has a characteristic fork head or "winged helix" DNA binding domain (DBD), has been implicated in the resumption of hookworm development in the host. However, the precise roles of Ac-DAF-16 in hookworm parasitism and its downstream targets are unknown. In the present study, we combined molecular techniques and bioinformatics to identify a group of Ac-DAF-16 binding sites and target genes.

Methodology/principal findings: The DNA binding domain of Ac-DAF-16 was used to select genomic fragments by in vitro genomic selection. Twenty four bound genomic fragments were analyzed for the presence of the DAF-16 family binding element (DBE) and possible alternative Ac-DAF-16 bind motifs. The 22 genes linked to these genomic fragments were identified using bioinformatics tools and defined as candidate direct gene targets of Ac-DAF-16. Their developmental stage-specific expression patterns were examined. Also, a new putative DAF-16 binding element was identified.

Conclusions/significance: Our results show that Ac-DAF-16 is involved in diverse biological processes throughout hookworm development. Further investigation of these target genes will provide insights into the molecular basis by which Ac-DAF-16 regulates its downstream gene network in hookworm infection.

Figure 1. Amino acid sequences of DNA binding domains from selected FOXO transcription factors were aligned using CLUSTAL W software on the San Diego Supercomputer Center Biology Workbench server (http://workbench.SDSC.edu) and displayed using BOXSHADE3.21 software located on Swiss EMBnet server (http://www.ch.embnet.org).

Identical amino acids are in red type, and conserved amino acids in blue. C-terminal arginine and lysine residues are shaded. The DBDs are from the following species: AcDAF16 (Ancylostoma caninum accession ACD85816); CeDAF16 (Caenorhabditis elegans, AAB84390); Foxo3, (Mus musculus, AAH19532); Foxo1 (Homo sapiens, AAH70065); Foxo4 (Homo sapiens, AAI06762).

Figure 2. Expression of Ac-DAF-16 DBD (aa 214–314) and Δ Ac-DAF-16 DBD (aa 220–292) in E. coli Rosetta (DE3) cells.

(A) Amino acid sequences of Ac-DAF-16 DBD and Δ Ac-DAF-16 DBD. Identical amino acids are in red. (B) Coomassie staining of purified rAc-DAF-16 DBD and rΔAc-DAF-16 DBD. (C) Western blot of rAc-DAF-16 DBD and rΔAc-DAF-16 DBD probed with anti-his (C-term) antibody. Lane 1, non-transformed Rosetta (DE3) cells; Lane 2, pET28a-Ac-DAF-16 DBD transformed Rosetta (DE3) cells in the absence of IPTG; Lane 3, pET28a-ΔAc-DAF-16 DBD transformed Rosetta (DE3) cells induced with IPTG; Lane 4, pET28a-Ac-DAF-16 DBD transformed Rosetta (DE3) cells induced with IPTG. The arrowheads indicate the position of Ac-DAF-16 DBD or Δ Ac-DAF-16 DBD.

Figure 3. Streptavidin bead pull-down to detect DBE binding activity of recombinant Ac-DAF-16 DBD and ΔAc-DAF-16 DBD.

Biotinylated dsDBE (Bio-DBE) was incubated with purified Ac-DAF-16 DBD or ΔAc-DAF-16 DBD, and peptide/oligonucleotides complexes were pulled down with streptavidin conjugated Sepharose beads. Precipitated DBD/oligo complexes were separated by SDS-PAGE and blotted to PVDF membranes for Western blotting using an anti-His (C-term) antibody. Bio-random represents a biotinylated oligomer of random sequence.

Figure 4. In vitro genomic selection of A. caninum DNA fragments containing Ac-DAF-16 binding sites.

(A–B) Immobilized rAc-DAF-16 DBD on Anti-FLAG M2 matrix confirmed by silver staining (A) and Western Blot with anti-FLAG antibody (B). The arrowheads indicate the position of Ac-DAF-16 DBD. M, protein standard; lane 1, Anti-FLAG M2 matrix; lane 2, anti-FLAG M2 matrix incubated with 2 ug of Ac-DAF-16 DBD and washed with 500 mM KCl. (C) A. caninum genomic DNA preparation. A 0.8% agarose gel was used to examine the DNA quality, and increasing amounts of a λ DNA standard were loaded to estimate DNA concentration. Lane 1–5, 30 ng λ DNA standard, 60 ng λ DNA standard, 90 ng λ DNA standard, 120 ng λ DNA standard, 150 ng λ DNA standard, respectively; lane 6, 2.5 µL of A. caninum genomic DNA sample; Lane7, 5 µL of A. caninum genomic DNA sample. (D) PCR amplification of the genomic fragments after each selection round. Lane 1, A. caninum genomic DNA sample cut with BfuCI; lane 2, 1st round purified PCR product; lane 3, 2nd round purified PCR product; lane 4, 3rd round purified PCR product; lane 5, 4th round purified PCR product.

Figure 5. Identification of a novel DAF-16 DBD binding element.

(A) Sequence logo of the putative DBD binding motif discovered using Gibbs Motif Sampler in bound fragments lacking a canonical DBE. (B) Streptavidin bead pull-down to detect the binding activity between positively selected genomic fragments and rAc-DAF-16 DBD. Biotin labeled PCR products were incubated with rAc-DAF-16 DBD. The protein/biotinylated PCR amplicon complexes were separated by SDS-PAGE and blotted to PVDF membrances for Western Blotting using anti-FLAG antibody. Lane1–4, Genome fragments from control selection; Lane 5, Fragment 3.23 (GACAAG motif); Lane 6, Fragment 3.28 (GACAAG motif); Lane 7, Fragment 4.6 (DBE); Lane 8, Fragment 2.23 (DBE).