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. 1998 Mar;66(3):1045-56.
doi: 10.1128/IAI.66.3.1045-1056.1998.

Enteric beta-defensin: molecular cloning and characterization of a gene with inducible intestinal epithelial cell expression associated with Cryptosporidium parvum infection

A P Tarver  1 , D P Clark, G Diamond, J P Russell, H Erdjument-Bromage, P Tempst, K S Cohen, D E Jones, R W Sweeney, M Wines, S Hwang, C L Bevins
Affiliations

Enteric beta-defensin: molecular cloning and characterization of a gene with inducible intestinal epithelial cell expression associated with Cryptosporidium parvum infection

A P Tarver et al. Infect Immun. 1998 Mar.

Erratum in

  • Infect Immun 1998 May;66(5):2399

Abstract

A growing body of evidence suggests that endogenous antibiotics contribute to the innate defense of mammalian mucosal surfaces. In the cow, beta-defensins constitute a large family of antibiotic peptides whose members have been previously isolated from the respiratory and oral mucosa, as well as circulating phagocytic cells. A novel bovine genomic clone with beta-defensin-related sequence [corrected] related to those of these alpha-defensins was isolated and characterized. The corresponding cDNA was isolated from a small intestinal library; its open reading frame predicts a deduced sequence of a novel beta-defensin, which we designate enteric beta-defensin (EBD). Northern blot analysis of a variety of bovine tissues revealed that EBD mRNA is highly expressed in the distal small intestine and colon, anatomic locations distinct from those for previously characterized beta-defensins. EBD mRNA was further localized by in situ hybridization to epithelial cells of the colon and small intestinal crypts. Infection of two calves with the intestinal parasite Cryptosporidium parvum induced 5- and 10-fold increases above control levels of EBD mRNA in intestinal tissues. An anchored-PCR strategy was used to identify other beta-defensin mRNAs expressed in the intestine. In addition to that of EBD, several low-abundance cDNAs which corresponded to other beta-defensin mRNAs were cloned. Most of these clones encoded previously characterized beta-defensins or closely related isoforms, but two encoded a previously uncharacterized prepro-beta-defensin. Northern blot evidence supported that all of these other beta-defensin genes are expressed at levels lower than that of the EBD gene in enteric tissue. Furthermore, some of these beta-defensin mRNAs were abundant in bone marrow, suggesting that in enteric tissue their expression may be in cells of hematopoietic origin. Extracts of small intestinal mucosa obtained from healthy cows have numerous active chromatographic fractions as determined by an antibacterial assay, and one peptide was partially purified. The peptide corresponded to one of the low-abundance cDNAs. This study provides evidence of beta-defensin expression in enteric tissue and that the mRNA encoding a major beta-defensin of enteric tissue, EBD, is inducibly expressed in enteric epithelial cells. These findings support the proposal that beta-defensins may contribute to host defense of enteric mucosa.

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Figures

FIG. 1
FIG. 1
Structure and sequence of the EBD gene. (A) Restriction map of the bovine EBD gene including 0.9 kb of 5′ flanking sequence. E, EcoRI; H, HindIII; P, PstI; UTR, untranslated region; SIG, signal. Also shown is a diagram of the predicted precursor structure of EBD deduced from the gene and cDNA sequences. (B) Nucleotide sequence of the EBD gene and alignment with the TAP gene sequence (13), with carets representing nucleotide identity. Exons (capital letters) were determined by comparison with EBD cDNA sequences (Fig. 2). Consensus sequences for TATA boxes (underlined), NF–IL-6 sites (boldface underlined), and H-APF-1 (double underlined) are indicated (see text). The consensus splice junction residues are shown in boldface. The polyadenylation signal is boxed.
FIG. 2
FIG. 2
cDNA sequence of EBD. (A) The upstream sequences of the individual RACE clones and the relevant portion of a lambda clone, BSI-13, are aligned with the genomic sequence (SEQ); colons denote sequence identity. The major transcription start site as determined by this analysis is indicated by the thick arrow, and the first uppercase nucleotide is designated nucleotide +1. Other possible transcription sites (−2, −3, and −47) are indicated by thin arrows. A consensus TATA box sequence is double underlined, and a second putative TATA box is single underlined. The presumed translation initiation codon, ATG, is boxed. (B) The cDNA sequence and the deducedprotein sequence of EBD are aligned with protein sequence of TAP. The position of the intron in the corresponding genomic sequence is indicated by a vertical line. The brackets in the TAP sequence encompass the isolated peptide found in tracheal tissues (15).
FIG. 3
FIG. 3
Southern blot hybridization analysis of the EBD gene. Bovine genomic DNA (10 μg) was digested with restriction endonucleases, and the products were size separated by agarose gel electrophoresis. The DNA was transferred to a nylon membrane and hybridized with 32P-end-labeled oligonucleotide EBD 285a (see Materials and Methods). Hybridization conditions were 5× SSC–1% SDS–5× Denhardt solution–40 μg of RNA per ml at 42°C in the presence of 37.5% (vol/vol) formamide. The high-stringency wash of the filter was with 2× SSC–0.1% SDS at 60°C for 60 min. The autoradiographic exposure was approximately 4 weeks. Numbers on the left indicate sizes (in basepairs) of mobility standards.
FIG. 4
FIG. 4
Northern blot analysis of EBD gene expression in bovine tissues. (A) Tissue distribution of β-defensins. Total RNA (20 μg) extracted from 14 different tissues was resolved by denaturing gel electrophoresis, capillary blotted to a nylon filter, and probed with either EBD 285a as an EBD probe, TAP286a as a TAP-specific probe, TAP48a as a common probe for β-defensins, or an α-tubulin probe. The hybridization and wash conditions were as described in Materials and Methods. Small intestine samples designated #1 and #2 represent RNAs extracted from the tissues of two healthy cows. (B) Expression of EBD in colonic tissue and usage of the putative upstream transcription start site. Total RNAs from the distal small (SM.) intestine, proximal colon (10 cm from the ileocecal junction), and distal colon (10 cm from the rectum) were analyzed as for panel A. The Northern blot was hybridized with EBD 285a to assess distribution of expression. The same blot was stripped of probe and rehybridized with EBD 9UTa, a probe from the unique sequence found in the 5′-extended RACE clone (Fig. 2, clone APT131.5) (see text). (C) Comparison of fetal and adult tissue expression of EBD. Total RNAs were isolated from the distal small intestine (S.I.) and colon of a bovine fetus at 4 months gestational age and from corresponding tissues of an adult cow and then analyzed as for panel A. (D) Northern blot analysis of EBD mRNA in enteric tissue from a calf infected with C. parvum. Total RNA was isolated from the distal 20 cm of small intestine from a C. parvum-infected calf and from a control uninfected calf. Analysis was as for panel A.
FIG. 5
FIG. 5
Detection of EBD mRNA by in situ hybridization. Paraffin-embedded sections of bovine ileum (A to D) and colon (E to H) were hybridized with EBD riboprobes labeled with [35S]UTP, washed under high-stringency conditions, and processed as described in Materials and Methods. (A, C, E, and G) Normal, uninfected intestinal tissue; (B, D, F, and H) intestinal tissue infected by C. parvum. The parasites are not visible at this magnification, but the blunting of the small intestinal villi and the inflammation of the lamina propria induced by the infection are easily seen in panels B and D. In normal ileum and colon, EBD mRNA is localized within epithelial cells at the base of the crypts (C and G). In C. parvum-infected ileum and colon, abundant EBD mRNA is present in epithelial cells throughout the elongated crypt (D and H). Magnification, ×80.
FIG. 6
FIG. 6
Northern blot analysis of several low-abundance β-defensin cDNAs cloned from small intestine by anchored PCR. Total RNAs (20 μg) extracted from 10 different tissues were resolved by denaturing gel electrophoresis, capillary blotted to a nylon filter, and probed with either JR335.B1 as a BNBD-9 probe, BNBD2/3-189a as an BNBD-3 probe, JR300.C7 as a BBD-C7 probe, EBD 285a as an EBD probe, or an α-tubulin probe. The hybridization and wash conditions were as for Fig. 4.
FIG. 7
FIG. 7
Isolation of β-defensin peptide from distal small intestine. An extract of bovine distal small intestine (200 g) was fractionated by using a combination of gel filtration and ion-exchange chromatography, and individual fractions were tested for antibacterial activity by an antibacterial plate assay (see Materials and Methods). (A) An active fraction from ion-exchange chromatography, eluting under conditions for previously characterized β-defensins, was applied to a C18 reverse-phase HPLC column and eluted with a gradient of acetonitrile (dashed line). The eluate was monitored by UV light absorbance (solid line), and each fraction (1 ml) was assayed for antibacterial activity (hash marks indicate active fractions). Fraction APT161G (arrow) was subjected to mass spectral analyses (see text) and N-terminal sequence analysis. (B) N-terminal sequence analysis. Two amino acids were identified at several positions, consistent with peptide heterogeneity within this fraction (see text). The mixed sequence is aligned with the sequences of TAP(S20N) (β-defensin) (54) and human group II phospholipase A2 (PLA2) (41).
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