Although lactoferrin has antimicrobial activity, its mechanism of action is not fully defined. Recently we have shown that the protein alters the Gram-negative outer membrane. As this membrane protects Gram-negative cells from lysozyme, we have studied whether lactoferrin's membrane effect could enhance the antibacterial activity of lysozyme. We have found that while each protein alone is bacteriostatic, together they can be bactericidal for strains of V. cholrae, S. typhimurium, and E. coli. The bactericidal effect is dose dependent, blocked by iron saturation of lactoferrin, and inhibited by high calcium levels, although lactoferrin does not chelate calcium. Using differing media, the effect of lactoferrin and lysozyme can be partially or completely inhibited; the degree of inhibition correlating with media osmolarity. Transmission electron microscopy shows that E. coli cells exposed to lactoferrin and lysozyme at 40 mOsm become enlarged and hypodense, suggesting killing through osmotic damage. Dialysis chamber studies indicate that bacterial killing requires direct contact with lactoferrin, and work with purified LPS suggests that this relates to direct LPS-binding by the protein. As lactoferrin and lysozyme are present together in high levels in mucosal secretions and neutrophil granules, it is probable that their interaction contributes to host defense. (J. Clin. Invest. 1991. 88:1080-1091

Many studies have shown that lactoferrin and transferrin have antimicrobial activity against gram-negative bacteria, but a mechanism of action has not been defined. We hypothesized that the iron-binding proteins could affect the gram-negative outer membrane in a manner similar to that of the chelator EDTA. The ability of lactoferrin and transferrin to release radiolabeled lipopolysaccharide (LPS) from a UDP-galactose epimerasedeficient Escherichia coli mutant and from wild-type Salmonella typhimurium strains was tested. Initial studies in barbital-acetate buffer showed that EDTA and lactoferrin cause significant release of LPS from all three strains. Further studies found that LPS release was blocked by iron saturation of lactoferrin, occurred between pH 6 and 7.5, was comparable for bacterial concentrations from 104 to 107 CFU/mI, and increased with increasing lactoferrin concentrations. Studies using Hanks balanced salt solution lacking calcium and magnesium showed that transferrin also could cause LPS release. Additionally, both lactoferrin and transferrin increased the antibacterial effect of a subinhibitory concentration of rifampin, a drug excluded by the bacterial outer membrane. This work demonstrates that these iron-binding proteins damage the gram-negative outer membrane and alter bacterial outer membrane permeability.

Iron-binding proteins have antibacterial activity; they have been identified in lung secretions, but their role in pulmonary antibacterial defenses is unclear. Murine lactoferrin and murine transferrin were used to generate polyclonal antiserum to lactoferrin and to transferrin, and the specificity of both antisera was shown by western blot. Mice were exposed to either aerosolized Escherichia coli or Staphylococcus aureus; they were killed 1, 4, 24, or 48 hr later; and their lungs were lavaged. We measured the levels of transferrin, lactoferrin, and albumin and did a cell count for the lavage fluid. The predominant iron-binding protein in resting animals was transferrin. Aerosolized E. coli caused a brisk PMNL response in the lungs that was associated with a major increase in the levels of lactoferrin. Challenge with S. aureus was associated with a moderate increase in the number of macrophages and a moderate decrease in the levels of transferrin and iron but no change in the levels of lactoferrin. The levels of iron-binding protein can vary according to the type of inflammatory response.