Beta-defensin-encoding genes are selected for divergent sequences of their mature bactericidal peptide and contribute to the immune defense against mastitis in the cow

Beta-defensins are bactericidal peptides serving as effector molecules of the innate immune system. They are synthesized in neutrophile granulocytes but also in a variety of epithelial tissue. The encoding genes form a multigene family in cattle comprising at least 13 members. We have sequenced the promoter and the entire gene encoding the [bêta]-Defensin BNBD5. The gene is segmented into two exons comprising 59 and 137 bp, respectively. Gene architecture, promoter and intron sequences are highly conserved among all [bêta]-Delensin-encoding genes known. However, the sequence of exon 2 - encoding the mature bactericidal peptide - reveals more sequence alterations than any other part of the gene. Moreover, we find that exon 2 sequences are positively selected for non-synonymous nucleotide exchanges leading to altered peptide sequences. The positive selection pressure favours apparently polarity differences, while maintaining the overall type of amino acids to conserve the protein structure. Differences in polarity might relate to different capabilities of the peptides to penetrate the bacterial cell wall. A phylogenetic analysis places the gene encoding the tracheal antibacterial peptide (TAP) into a separate basal position of [bêta]-Defensin gene evolution while the other genes form distinct paired groups indicating recent events of gene duplication. We have analysed some aspects of promoter function and tissue specific gene expression of the BNBD5-encoding gene. Primer extension analyses demonstrate at least three strong transcriptional start sites, each preceeded by a TATA-box. Real-time PCR reveals that this gene is strongly expressed in lung and lactating mammary gland, poorly in adipose tissue, but not in liver. Mastitis infection induces gene expression strongly (>15 fold) in some but not in all cases. In-situ hybridizations localize the Mammary Epithelial Cell (NEC) as site of expression in the lactating mammary gland. They reveal in sections of apparently healthy udders a pattern of regionally restricted gene induction. Moreover, not all MEC of a given alveolum are eventually induced, but only some adjacent cells. This differs from the regulation of caseine-gene-expression, in which all MEC of an alveolum are regulated in concert. Rather, our in-situ hybridizations indicate a locally restricted, non-systemic induction of this [bêta]-Defensin-encoding gene in the udder. Our data together proove that the BNBD5 -encoding gene is a candidate gene for mastitis protection. They support the concept that bacteria contacting the epithelial cells induce BNBD5 expression directly in these cells. A 1093 bp comprising segment of the promoter of the BNBD5-encoding gene drives strongly the expression of a luciferase reporter gene in transiently transfected murine HC-11 MEC. Expression is significantly induced (~4 fold), if the cells are incubated with heat inactivated E. coli (strain XL1-blue, 107 cells /ml). Truncation of the promoter down to position - 445 leaves the inducibility unchanged, albeit deleting two functional (EMSA-supershift analyses) and evolutionary conserved STAT5-binding sites (at positions -854 and -744). However, inducibility is severely impaired if the promoter is truncated down to positions -282 (1.8 fold) or -68 (1.5 fold). Interestingly, the -282 construct harbors still the NF-[kappa]B attachment site, located downstream of position -272. NF-[kappa]B is known to be a key player in Toll-like receptor-mediated gene induction. (Texte intégral