Antimicrobial activity of murine lung cells against Staphylococcus aureus is increased in vitro and in vivo after elafin gene transfer.

Staphylococcus aureus is a pathogen often found in pneumonia and sepsis. In the context of the resistance of this organism to conventional antibiotics, an understanding of the regulation of natural endogenous antimicrobial molecules is of paramount importance. Previous studies have shown that both human and mouse airways express a variety of these molecules, including defensins, cathelicidins, and the four-disulfide core protein secretory leukocyte protease inhibitor. We demonstrate here by culturing mouse tracheal epithelial cells at an air-liquid interface that, despite the production of Defb1, Defb14, and Defr1 in this system, these cells are unable to clear S. aureus when exposed to this respiratory pathogen. Using an adenovirus (Ad)-mediated gene transfer strategy, we show that overexpression of elafin, an anti-elastase/antimicrobial molecule (also a member of the four-disulfide core protein family), dramatically improves the clearance of S. aureus. In addition, we also demonstrate that this overexpression is efficient in vivo and that intratracheal instillation of Ad-elafin significantly reduced the lung bacterial load and demonstrates concomitant anti-inflammatory activity by reducing neutrophil numbers and markers of lung inflammation, such as bronchoalveolar lavage levels of tumor necrosis factor and myeloperoxidase. These findings show that an increased antimicrobial activity phenotype is provided by the elafin molecule and have implications for its use in S. aureus-associated local and systemic infections.

Rapid sequence divergence in mammalian beta-defensins by adaptive evolution.

beta-Defensin genes encode broad spectrum antimicrobial cationic peptides. We have analysed the largest murine and human clusters of these genes, which localise to mouse and human chromosome 8. Using hidden Markov models, we identified novel mouse and human beta-defensin genes. We subsequently found full-length expressed transcripts for these novel genes. Expression in the mouse was high in brain and reproductive tissues. Fourteen murine beta-defensins could be grouped into two clear sub-groups by virtue of their position and high signal sequence (exon 1 encoded) identity. In contrast, there was a very low level of sequence conservation in the exon 2 region encoding the mature antimicrobial peptide. Evolutionary analysis revealed strong evidence that following gene duplication, exon 1 and surrounding non-coding DNA show little divergence within subfamilies. The focus for rapid sequence divergence is localised in the DNA encoding the mature peptide and this is driven by accelerated positive selection. In the human we also conclude that the locus has evolved by successive rounds of duplication followed by substantial divergence involving positive selection, to produce a diverse cluster of paralogous genes prior to human-baboon divergence. This mechanism of adaptive evolution is consistent with the role of this gene family as defence against bacterial pathogens. In order to look at function of these rapidly evolving genes, we characterised one of the novel mouse beta-defensin genes. This gene deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta defensins. This defensin related gene (Defr1) is most highly expressed in testis and heart and the genomic organisation is highly similar to Defb3-6. A synthetic Defr1 peptide was shown to exist as a dimer and yet displayed both antimicrobial and chemotactic activity. The antimicrobial activity of Defr1 against S. aureus, E. coli and B. cepacia was found to be reduced in raised concentration of NaCl, but its action against P. aeruginosa was independent of NaCl concentration. These data have major implications on the structure and functions of these important host defence molecules.

Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens.

Elafin (elastase-specific inhibitor) is a low molecular weight inhibitor of neutrophil elastase which is secreted in the lung. Using synthetic peptides corresponding to full-length elafin (H2N-1AVT.....95Q-OH), the NH2-terminal domain (H2N-1AVT.....50K-OH) and the COOH-terminal domain (H2N-51PGS.....95Q-OH), we demonstrate that elafin's anti-elastase activity resides exclusively in the COOH-terminus. Several characteristics of elafin suggest potential anti-microbial activity. The anti-microbial activity of elafin, and of its two structural domains, was tested against the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Elafin killed both bacteria efficiently, with 93% killing of P. aeruginosa by 2.5 microM elafin and 48% killing of S. aureus by 25 microM elafin. For both organisms, full-length elafin was required to optimise bacterial killing. These findings represent the first demonstration of co-existent anti-proteolytic and anti-microbial functions for elafin.

Mouse beta defensin-1 is a functional homolog of human beta defensin-1.

Defensin are 3-4 kDa antimicrobial peptides of which three distinct families have been identified; alpha-defensin, beta-defensins, and insect defensins. Recent investigations have shown that beta-defensins are present in the human airways and may be relevant to the pathogenesis of cystic fibrosis (CF) lung disease. We report here the further characterization of a recently identified mouse beta-defensin gene, Defb1, sometimes referred to as mBD-1, which is homologous to the human airway beta defensin hBD-1. We report that Defb1 is expressed in a variety of tissues including the airways and, similar to hBD-1, is not upregulated by lipopolysaccharide (LPS). Defb1 was found to consist of two small exons separated by a 16-kb intron and cytogenetic, and physical mapping linked it to the alpha defensin gene cluster on mouse Chromosome (Chr) 8. Functional studies demonstrate that, like hBD-1, Defb1 demonstrates a salt-sensitive antimicrobial activity against Pseudomonas aeruginosa. Of relevance to CF lung disease is the fact that neither the hBD-1 nor the mBD-1 peptides are active against Burkholderia cepacia.