Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactofenin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.

Incubation of Escherichia coli (104 organisms per ml) in cell-free rabbit lung lavage for 30 min at 37°C resulted in a 70% reduction in colony counts on deoxycholate agar. A low-molecular-weight peptide (about 3,400 daltons), with zinc as a cofactor, was responsible for this activity. The peptide was isolated by Sephadex G-15 separation of lyophilized rabbit lung lavage which had been centrifuged to remove macrophages and suspended phosphlipids and passed through a 10,000-dalton (pore size) membrane filter. Peptide activity against E. coli was inhibited by phosphate buffer but not by borate, Tris, or barbital buffer. Bacteria incubated in phosphate buffer and then washed in saline were resistant to peptide activity. Antibacterial activity was also inhibited when peptide-exposed bacteria were incubated in phosphate buffer before deoxycholate treatment. 32P-radiolabeled E. coli cells lost about 20% of their radiolabel after 15 min of incubation with peptide.

The contribution of extracellular secretions to the antibacterial defenses of the lungs remains poorly defined. Recent studies have demonstrated that mouse and rabbit bronchoalveolar washings contain a low-molecular-weight peptide that has antibacterial activity against Escherichia coli. In this study we investigated whether a similar peptide could be identified in human secretions. Bronchoalveolar lavage fluid was obtained from normal volunteers and patients with interstitial lung disease or pulmonary alveolar proteinosis. Cellular material and surfactant lipids were removed from the fluid by sequential centrifugations, and the supernatant was fractionated by exclusion filtration to isolate peptides with a molecular weight less than 10,000. Gel filtration chromatography separated the ultrafiltrate into several peaks, the first of which had antibacterial activity against E. coli. This material was further separated into several hydrophilic peaks by reverse-phase high-pressure liquid chromatography (RPHPLC). All samples had similar RPHPLC graphs. Material from the third RPHPLC peak produced an antibacterial effect similar to that produced by the rabbit and mouse peptide.

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