Activity, expression and genetic variation of canine β-defensin 103: a multifunctional antimicrobial peptide in the skin of domestic dogs

Abstract

The skin functions as more than a physical barrier to infection. Epithelial cells of the skin can synthesize antimicrobial peptides, including defensins, which exhibit direct antimicrobial activity. Here we characterize the expression pattern, genetic variation and activity of the major β-defensin expressed in canine skin, canine β-defensin 103 (CBD103). The gene encoding CBD103 exhibits two forms of polymorphism: a common 3-basepair deletion allele and a gene copy-number variation. Golden retrievers and Labrador retrievers were the only breeds that encoded the variant allele of CBD103, termed CBD103ΔG23. Both these breeds also exhibited a CBD103 gene copy-number polymorphism that ranged from 2 to 4 gene-copies per diploid genome. Recombinant CBD103 and CBD103ΔG23, as well as the human ortholog human β-defensin 3 (hBD3) and hBD3ΔG23, showed potent and comparable antimicrobial killing against both methicillin-susceptible and methicillin-resistant Staphylococcus pseudintermedius. Skin biopsy specimens from dogs with atopic dermatitis revealed CBD103 expression levels similar to those in healthy controls and comparable at lesional and nonlesional sites. This expression pattern in dogs differs from the previously reported reduced expression of the human ortholog in atopic dermatitis. Overall, the similarities of CBD103 and its human ortholog reported here support the notion that the domestic dog may serve as a valuable model for studying β-defensin biology in the skin.

Fig. 1

Expression of canine β-defensins. qRT-PCR analysis of absolute copy number of mRNA transcripts encoding CBD1 (a) and CBD103 (b) in RNA isolated from various tissues including the skin, gastrointestinal tract (tongue, stomach, duodenum, jejunum, ileum, colon, rectum), lungs, kidneys, pancreas, bone marrow, testes and epididymis. Each bar represents an average of replicate assays (standard deviation <10%) from a single specimen tested (n = 1). ND = Not detected.

Fig. 2

Epidermal mRNA and peptide expression of CBD103. a qRT-PCR analysis of absolute copy number of mRNA transcripts encoding CBD103 in RNA isolated from the caudodorsal (dorsal) and inguinal (ventral) skin of dogs. Calculated mean of the 6 dogs, dorsal and ventral skin. Error bars represent standard error. b Immunostaining of CBD103 in canine skin (2×) using a polyclonal antibody (1:500) raised against hBD3. CBD103 localizes to keratinocytes and follicular epithelial cells. Inset is taken at 20×. c Negative control for staining with secondary antibody alone (2×). Inset is taken at 20×. Arrowheads indicate positive staining. Size bar in 2× view: 300 µm, and in 20× view: 30 µm.

Fig. 3

Prevalence of CBD103 common variant and gene copy-number variation. a Pie graphs represent individuals of a given breed, divided into canine genotypes: CBD103/CBD103 (blue), CBD103/CBD103ΔG23 (yellow) and CBD103ΔG23/CBD103ΔG23 (red). Doberman Pinschers (n = 15), German Shepherds (n = 15) and Rottweilers (n = 15) do not appear to have the CBD103ΔG23 allele, whereas Golden retrievers (n = 13) and Labrador retrievers (n = 16) possess the CBD103ΔG23 allele as a heterozygote or homozygote variant. b Plot of CBD103 gene copy-number variation with colors indicating the CBD103 genotype. Doberman Pinschers, German Shepherds and Rottweilers encode 2 copies of CBD103 per diploid genome, whereas Golden retrievers and Labrador retrievers have variation in their total CBD103 gene copy number.

Fig. 4

CBD103 and hBD3 peptide sequence comparison and recombinant expression. a Predicted mature peptide sequences for CBD103 and CBD103ΔG23, as well as hBD3 and hBD3ΔG23. Asterisks designate amino acid differences, all of which are conservative except S37G and T58R. b AU-PAGE of TCEP reduced recombinant CBD103, CBD103ΔG23, hBD3 and hBD3ΔG23 showing peptide homogeneity by SimplyBlue staining. c Western blot of TCEP reduced recombinant CBD103, CBD103ΔG23, hBD3 and hBD3ΔG23 using a polyclonal antibody (1:3,000) raised against hBD3.

Fig. 5

Antibacterial activity of recombinant CBD103. Determination of antibacterial activity of CBD103, CBD103ΔG23, hBD3 and hBD3ΔG23 using a radial diffusion antibacterial assay with clinical isolates of methicillin-susceptible (a) and methicillin-resistant (b) S. pseudintermedius. Graphical representation of the antibacterial clearing by CBD103, CBD103ΔG23, hBD3 and hBD3ΔG23 [30].

Fig. 6

CBD103 expression in AD. Quantitative RT-PCR analysis of absolute copy-number of mRNA transcripts encoding RPS5, CBD103 and S100A8 in RNA isolated from lesional and nonlesional skin biopsy specimens from dogs (n = 18) diagnosed with AD. Error bars represent standard error.