Mapping and sequencing of the canine NRAMP1 gene and identification of mutations in leishmaniasis-susceptible dogs

Abstract

The NRAMP1 gene (Slc11a1) encodes an ion transporter protein involved in the control of intraphagosomal replication of parasites and in macrophage activation. It has been described in mice as the determinant of natural resistance or susceptibility to infection with antigenically unrelated pathogens, including Leishmania. Our aims were to sequence and map the canine Slc11a1 gene and to identify mutations that may be associated with resistance or susceptibility to Leishmania infection. The canine Slc11a1 gene has been mapped to dog chromosome CFA37 and covers 9 kb, including a 700-bp promoter region, 15 exons, and a polymorphic microsatellite in intron 1. It encodes a 547-amino-acid protein that has over 87% identity with the Slc11a1 proteins of different mammalian species. A case-control study with 33 resistant and 84 susceptible dogs showed an association between allele 145 of the microsatellite and susceptible dogs. Sequence variant analysis was performed by direct sequencing of the cDNA and the promoter region of four unrelated beagles experimentally infected with Leishmania infantum to search for possible functional mutations. Two of the dogs were classified as susceptible and the other two were classified as resistant based on their immune responses. Two important mutations were found in susceptible dogs: a G-rich region in the promoter that was common to both animals and a complete deletion of exon 11, which encodes the consensus transport motif of the protein, in the unique susceptible dog that needed an additional and prolonged treatment to avoid continuous relapses. A study with a larger dog population would be required to prove the association of these sequence variants with disease susceptibility.

FIG. 1.

Genomic structure of the canine Slc11a1 gene. nt, nucleotides.

FIG. 2.

Neighbor-joining relationship tree of mammalian Slc11 cDNA sequences. The distance indicates the number of nucleotide differences. Numbers on the nodes are percent bootstrap values from 1,000 replications, and a scale bar for branch lengths is shown.

FIG. 3.

Sequence analysis. (A) Promoter and 5′ UTR of the canine Slc11a1 gene from nucleotide 1 to the translational start codon (position 689). Putative binding sites for transcription factors are indicated with a line above (sense strand) or below (antisense strand) the consensus sequence. (B) G-rich polymorphism of the promoter regions from beagles and a Rottweiler. GM-CSF, granulocyte-macrophage colony-stimulating factor.

FIG. 4.

Slc11a1 mapped to canine RH syntenic group 11.

FIG. 5.

Allelic frequencies of the intron 1 microsatellite. x axis, basepairs of alleles; y axis, allelic frequency.

FIG. 6.

mRNA alternative splicing in dog S1. (a) PCR amplification from exon 9 to the 3′ UTR of cDNAs from the four beagles experimentally infected with L. infantum. R1 and R2 are resistant dogs, and S1 and S2 are susceptible dogs. (b) PCR amplification of the same region of dog S1 from three different RNA extractions. Two independent RT-PCRs of each RNA extraction are shown (lanes 1 and 2). Lane M1, φX174 HaeIII; M2, 1-kb ladder.

FIG. 7.

Multiple alignment of the Slc11a1 proteins from the Rottweiler (ROTW.) and the four beagles experimentally infected with L. infantum. For dog S1, the normal allele is shown. TM domains are indicated by thin overlining, and the consensus transport motif of the protein is indicated by thick overlining. Potential N-glycosylation sites are boxed, and potential phosphorylation sites for protein kinase C are indicated by double overlining. Exons are separated by vertical lines.