Cloning and Characterization of the Gene for a New Epithelial β-Defensin

Abstract

Mammalian ␤-defensins are endogenous cysteine-rich peptide antibiotics that are produced either by epithelial cells lining the respiratory, digestive, and urogenital tracts or by granulocytes and macrophages. A growing body of evidence has implicated these peptides in host defense, particularly mucosal innate immunity. We previously reported the cloning of the full-length cDNA for a porcine ␤-defensin (pBD-1), which was found to be expressed throughout the airway and oral mucosa. Here, we provide the structural organization of the pBD-1 gene, showing that the entire gene spans ϳ1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Fluorescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15.1 within a region of conserved synteny to the chromosomal locations of human and mouse ␣-and ␤-defensins. We also provide several independent lines of evidence showing that the pBD-1 gene is expressed constitutively during inflammation and infection, despite its resemblance to many inducible epithelial ␤-defensins in amino acid sequence, genomic structure, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-␣, and interleukin (IL)-1␤ failed to up-regulate the expression of pBD-1 mRNA. Second, pBD-1 gene expression was not enhanced in either digestive or respiratory mucosa of pigs following a 2-day infection with Salmonella typhimurium or Actinobacillus pleuropneumoniae. Last, direct transfection of the pBD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1␤. The constitutive expression of pBD-1 in airway and oral mucosa, which is consistent with a lack of consensus binding sites for nuclear factor-B or NF-IL-6 in its promoter region, suggests that it may play a surveillance role in maintaining the steady state of microflora on mucosal surfaces.

Modulation of Porcine β-Defensins 1 and 2 upon Individual and Combined Fusarium Toxin Exposure in a Swine Jejunal Epithelial Cell Line

“…Zhang et al (8) and Elahi et al (33) previously showed pBD-1 to be constitutively expressed in different tissues (8). However, induction of pBD-1 mRNA expression may also be stimulated upon exposure to food contaminants or enteric pathogens (10,33,34) and is in line with our data, which show pBD-1 mRNA expression was upregulated following exposure to DON, NIV, ZEA, and FB1, individually and in mixtures. Elevated expression of pBD-1 in, for example, IECs may play a potential role in surveillance and maintenance of a homeostatic state of microflora on the mucosal epithelium and may be significant in preventing the development or progression of diseases (33,35,36).…”

Gene Silencing and Overexpression of Porcine Peptidoglycan Recognition Protein Long Isoforms: Involvement in β-Defensin-1 Expression

“…Gene-specific primers for pPGRP-L1 were sense, 5Ј-CTG TGG TAG CAG CAG CTT CTC TTT-3Ј, and antisense, 5Ј-GCT GCA GAA GCA ATC CAT ACA GGA-3Ј (cDNA nucleotides 12 to 35 and 256 to 233), and those for pPGRP-L2 were sense, 5Ј-AGC CCA GAG TTT CAA GGC CTG ATT-3Ј, and antisense, 5Ј-TCA GGA ACT GTG GCT CCA ATG TCT-3Ј (cDNA nucleotides 368 to 391 and 602 to 579). Primers for ␤-defensin-1 were as described previously (48). DNase-treated total RNA (500 ng) was used in each 25-l RT-PCR mixture.…”

“…After amplification, 10 l of each reaction mixture was analyzed by 2% agarose gel electrophoresis, and bands were visualized by ethidium bromide staining. For Southern analysis, PCR products were transferred onto Hybond Nϩ filters (Amersham Pharmacia Biotech), hybridized with PGRP cDNA probes labeled with [␣-32 P]dCTP (3,000 Ci/mmol), and detected using autoradiography as previously described (48).…”

Antimicrobial Characterization of Site-Directed Mutagenesis of Porcine Beta Defensin 2

“…Relatively little work have been done on porcine defensins and they have not yet been isolated from natural sources [7, 9–10]. pBD1, the first member of porcine β defensin, has a strong inhibitory activity against gram-negative bacteria [11–12]. On the contrary, porcine β defensin 2 (pBD2) shows strong antibacterial activity against both gram-negative and gram-positive bacteria, including multi-resistant bacteria.…”