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Logo of jcinvestThe Journal of Clinical Investigation
J Clin Invest. 1991 October; 88(4): 1080–1091.
PMCID: PMC295557

Killing of gram-negative bacteria by lactoferrin and lysozyme.


Although lactoferrin has antimicrobial activity, its mechanism of action is not full 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. cholerae, 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.

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Selected References

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  • Bullen JJ, Rogers HJ, Griffiths E. Role of iron in bacterial infection. Curr Top Microbiol Immunol. 1978;80:1–35. [PubMed]
  • Cohen MS, Britigan BE, French M, Bean K. Preliminary observations on lactoferrin secretion in human vaginal mucus: variation during the menstrual cycle, evidence of hormonal regulation, and implications for infection with Neisseria gonorrhoeae. Am J Obstet Gynecol. 1987 Nov;157(5):1122–1125. [PubMed]
  • Wright DG, Gallin JI. Secretory responses of human neutrophils: exocytosis of specific (secondary) granules by human neutrophils during adherence in vitro and during exudation in vivo. J Immunol. 1979 Jul;123(1):285–294. [PubMed]
  • Bullen JJ. The significance of iron in infection. Rev Infect Dis. 1981 Nov-Dec;3(6):1127–1138. [PubMed]
  • Iacono VJ, MacKay BJ, DiRienzo S, Pollock JJ. Selective antibacterial properties of lysozyme for oral microorganisms. Infect Immun. 1980 Aug;29(2):623–632. [PMC free article] [PubMed]
  • Jollès P, Jollès J. What's new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biochem. 1984 Sep;63(2):165–189. [PubMed]
  • Leive L. The barrier function of the gram-negative envelope. Ann N Y Acad Sci. 1974 May 10;235(0):109–129. [PubMed]
  • Nikaido H, Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. [PMC free article] [PubMed]
  • Martinez RJ, Carroll SF. Sequential metabolic expressions of the lethal process in human serum-treated Escherichia coli: role of lysozyme. Infect Immun. 1980 Jun;28(3):735–745. [PMC free article] [PubMed]
  • Masson PL, Heremans JF, Prignot JJ, Wauters G. Immunohistochemical localization and bacteriostatic properties of an iron-binding protein from bronchial mucus. Thorax. 1966 Nov;21(6):538–544. [PMC free article] [PubMed]
  • Finkelstein RA, Sciortino CV, McIntosh MA. Role of iron in microbe-host interactions. Rev Infect Dis. 1983 Sep-Oct;5 (Suppl 4):S759–S777. [PubMed]
  • Spik G, Cheron A, Montreuil J, Dolby JM. Bacteriostasis of a milk-sensitive strain of Escherichia coli by immunoglobulins and iron-binding proteins in association. Immunology. 1978 Oct;35(4):663–671. [PubMed]
  • Stephens S, Dolby JM, Montreuil J, Spik G. Differences in inhibition of the growth of commensal and enteropathogenic strains of Escherichia coli by lactotransferrin and secretory immunoglobulin A isolated from human milk. Immunology. 1980 Nov;41(3):597–603. [PubMed]
  • Arnold RR, Cole MF, McGhee JR. A bactericidal effect for human lactoferrin. Science. 1977 Jul 15;197(4300):263–265. [PubMed]
  • Ellison RT, 3rd, Giehl TJ, LaForce FM. Damage of the outer membrane of enteric gram-negative bacteria by lactoferrin and transferrin. Infect Immun. 1988 Nov;56(11):2774–2781. [PMC free article] [PubMed]
  • Ellison RT, 3rd, Luo Q, Reller LB. Enhancement of the activity of cefotaxime by iron-binding proteins. J Antimicrob Chemother. 1990 Mar;25(3):479–481. [PubMed]
  • Ellison RT, 3rd, LaForce FM, Giehl TJ, Boose DS, Dunn BE. Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+. J Gen Microbiol. 1990 Jul;136(7):1437–1446. [PubMed]
  • Vaara M, Vaara T. Polycations as outer membrane-disorganizing agents. Antimicrob Agents Chemother. 1983 Jul;24(1):114–122. [PMC free article] [PubMed]
  • Hancock RE, Wong PG. Compounds which increase the permeability of the Pseudomonas aeruginosa outer membrane. Antimicrob Agents Chemother. 1984 Jul;26(1):48–52. [PMC free article] [PubMed]
  • Lehrer RI, Barton A, Daher KA, Harwig SS, Ganz T, Selsted ME. Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J Clin Invest. 1989 Aug;84(2):553–561. [PMC free article] [PubMed]
  • Weiss J, Muello K, Victor M, Elsbach P. The role of lipopolysaccharides in the action of the bactericidal/permeability-increasing neutrophil protein on the bacterial envelope. J Immunol. 1984 Jun;132(6):3109–3115. [PubMed]
  • West SE, Sparling PF. Response of Neisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production. Infect Immun. 1985 Feb;47(2):388–394. [PMC free article] [PubMed]
  • Osserman EF, Lawlor DP. Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med. 1966 Nov 1;124(5):921–952. [PMC free article] [PubMed]
  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. [PubMed]
  • MCGILVERY RW, MOKRASCH LC. Purification and properties of fructose-1, 6-diphosphatase. J Biol Chem. 1956 Aug;221(2):909–917. [PubMed]
  • Blaser MJ, Hopkins JA, Berka RM, Vasil ML, Wang WL. Identification and characterization of Campylobacter jejuni outer membrane proteins. Infect Immun. 1983 Oct;42(1):276–284. [PMC free article] [PubMed]
  • Oakley BR, Kirsch DR, Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. [PubMed]
  • Perez Perez GI, Blaser MJ. Lipopolysaccharide characteristics of pathogenic campylobacters. Infect Immun. 1985 Feb;47(2):353–359. [PMC free article] [PubMed]
  • Hitchcock PJ. Aberrant migration of lipopolysaccharide in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Eur J Biochem. 1983 Jul 1;133(3):685–688. [PubMed]
  • Goldman RC, Leive L. Heterogeneity of antigenic-side-chain length in lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2. Eur J Biochem. 1980;107(1):145–153. [PubMed]
  • Hukari R, Helander IM, Vaara M. Chain length heterogeneity of lipopolysaccharide released from Salmonella typhimurium by ethylenediaminetetraacetic acid or polycations. Eur J Biochem. 1986 Feb 3;154(3):673–676. [PubMed]
  • Wilkinson RG, Gemski P, Jr, Stocker BA. Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J Gen Microbiol. 1972 May;70(3):527–554. [PubMed]
  • Joiner KA, Goldman R, Schmetz M, Berger M, Hammer CH, Frank MM, Leive L. A quantitative analysis of C3 binding to O-antigen capsule, lipopolysaccharide, and outer membrane protein of E. coli 0111B4. J Immunol. 1984 Jan;132(1):369–375. [PubMed]
  • Vaara M, Vaara T. Polycations sensitize enteric bacteria to antibiotics. Antimicrob Agents Chemother. 1983 Jul;24(1):107–113. [PMC free article] [PubMed]
  • Sanderson KE, Ross H, Ziegler L, Mäkelä PH. F + , Hfr, and F' strains of Salmonella typhimurium and Salmonella abony. Bacteriol Rev. 1972 Dec;36(4):608–637. [PMC free article] [PubMed]
  • Dalmastri C, Valenti P, Visca P, Vittorioso P, Orsi N. Enhanced antimicrobial activity of lactoferrin by binding to the bacterial surface. Microbiologica. 1988 Jul;11(3):225–230. [PubMed]
  • Schryvers AB. Identification of the transferrin- and lactoferrin-binding proteins in Haemophilus influenzae. J Med Microbiol. 1989 Jun;29(2):121–130. [PubMed]
  • Lee BC, Bryan LE. Identification and comparative analysis of the lactoferrin and transferrin receptors among clinical isolates of gonococci. J Med Microbiol. 1989 Mar;28(3):199–204. [PubMed]
  • Wollenweber HW, Morrison DC. Synthesis and biochemical characterization of a photoactivatable, iodinatable, cleavable bacterial lipopolysaccharide derivative. J Biol Chem. 1985 Dec 5;260(28):15068–15074. [PubMed]
  • Brade L, Brandenburg K, Kuhn HM, Kusumoto S, Macher I, Rietschel ET, Brade H. The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment. Infect Immun. 1987 Nov;55(11):2636–2644. [PMC free article] [PubMed]
  • Labischinski H, Barnickel G, Bradaczek H, Naumann D, Rietschel ET, Giesbrecht P. High state of order of isolated bacterial lipopolysaccharide and its possible contribution to the permeation barrier property of the outer membrane. J Bacteriol. 1985 Apr;162(1):9–20. [PMC free article] [PubMed]
  • Lehrer RI, Ganz T. Antimicrobial polypeptides of human neutrophils. Blood. 1990 Dec 1;76(11):2169–2181. [PubMed]
  • Curtis NA, Eisenstadt RL, East SJ, Cornford RJ, Walker LA, White AJ. Iron-regulated outer membrane proteins of Escherichia coli K-12 and mechanism of action of catechol-substituted cephalosporins. Antimicrob Agents Chemother. 1988 Dec;32(12):1879–1886. [PMC free article] [PubMed]
  • Watanabe NA, Nagasu T, Katsu K, Kitoh K. E-0702, a new cephalosporin, is incorporated into Escherichia coli cells via the tonB-dependent iron transport system. Antimicrob Agents Chemother. 1987 Apr;31(4):497–504. [PMC free article] [PubMed]
  • Valenti P, Visca P, Antonini G, Orsi N, Antonini E. The effect of saturation with Zn2+ and other metal ions on the antibacterial activity of ovotransferrin. Med Microbiol Immunol. 1987;176(3):123–130. [PubMed]
  • Anderson BF, Baker HM, Norris GE, Rice DW, Baker EN. Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. J Mol Biol. 1989 Oct 20;209(4):711–734. [PubMed]
  • Bennett RM, Bagby GC, Davis J. Calcium-dependent polymerization of lactoferrin. Biochem Biophys Res Commun. 1981 Jul 16;101(1):88–95. [PubMed]
  • NEWTON BA. Site of action of polymyxin on Pseudomonas aeruginosa: antagonism by cations. J Gen Microbiol. 1954 Jun;10(3):491–499. [PubMed]
  • Zimelis VM, Jackson GG. Activity of aminoglycoside antibiotics aganst Pseudomonas aeruginosa: specificity and site of calcium and magnesium antagonism. J Infect Dis. 1973 Jun;127(6):663–669. [PubMed]
  • Mannion BA, Weiss J, Elsbach P. Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli. J Clin Invest. 1990 Mar;85(3):853–860. [PMC free article] [PubMed]
  • Weiss J, Victor M, Elsbach P. Role of charge and hydrophobic interactions in the action of the bactericidal/permeability-increasing protein of neutrophils on gram-negative bacteria. J Clin Invest. 1983 Mar;71(3):540–549. [PMC free article] [PubMed]
  • Weiss J, Franson C, Schmeidler K, Elsbach P. Reversible envelope effects during and after killing of Escherichia coli w by a highly-purified rabbit polymorpho-nuclear leukocyte fraction. Biochim Biophys Acta. 1976 Jun 4;436(1):154–169. [PubMed]
  • Bezwoda WR, Mansoor N. Lactoferrin from human breast milk and from neutrophil granulocytes. Comparative studies of isolation, quantitation, characterization and iron binding properties. Biomed Chromatogr. 1989 May;3(3):121–126. [PubMed]
  • Anderson BF, Baker HM, Norris GE, Rumball SV, Baker EN. Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins. Nature. 1990 Apr 19;344(6268):784–787. [PubMed]
  • Arnold RR, Brewer M, Gauthier JJ. Bactericidal activity of human lactoferrin: sensitivity of a variety of microorganisms. Infect Immun. 1980 Jun;28(3):893–898. [PMC free article] [PubMed]
  • Bortner CA, Miller RD, Arnold RR. Bactericidal effect of lactoferrin on Legionella pneumophila. Infect Immun. 1986 Feb;51(2):373–377. [PMC free article] [PubMed]
  • Mathur NB, Dwarkadas AM, Sharma VK, Saha K, Jain N. Anti-infective factors in preterm human colostrum. Acta Paediatr Scand. 1990 Nov;79(11):1039–1044. [PubMed]
  • Thompson AB, Bohling T, Payvandi F, Rennard SI. Lower respiratory tract lactoferrin and lysozyme arise primarily in the airways and are elevated in association with chronic bronchitis. J Lab Clin Med. 1990 Feb;115(2):148–158. [PubMed]
  • Pollack C, Straley SC, Klempner MS. Probing the phagolysosomal environment of human macrophages with a Ca2+-responsive operon fusion in Yersinia pestis. Nature. 322(6082):834–836. [PubMed]

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