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Infection and Immunity, September 2009, p. 4028-4040, Vol. 77, No. 9
0019-9567/09/$08.00+0     doi:10.1128/IAI.00232-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Human {alpha}-Defensins Inhibit Hemolysis Mediated by Cholesterol-Dependent Cytolysins{triangledown}

Robert I. Lehrer,1* Grace Jung,1 Piotr Ruchala,1 Wei Wang,1,2 Ewa D. Micewicz,1 Alan J. Waring,1 Eugene J. Gillespie,3 Kenneth A. Bradley,3 Adam J. Ratner,4 Richard F. Rest,5 and Wuyuan Lu6

Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California 90095,1 Amgen, Thousand Oaks, California,2 Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095,3 Departments of Pediatrics and Microbiology, Columbia University, New York, New York 10032,4 Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129,5 Institute for Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 212016

Received 26 February 2009/ Returned for modification 7 April 2009/ Accepted 24 June 2009

Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependent cytolysins. Here, we report that human {alpha}-defensins HNP-1 to HNP-3 acted in a concentration-dependent manner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO), listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against the other toxins. Human {alpha}-defensins HNP-4 and HD-6 and human β-defensin-1, -2, and -3 lacked protective ability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog, in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced binding and protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Abu residues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmon resonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also that solution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibited hemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or to cholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B of Clostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first time these defensins have been shown to inhibit pore-forming toxins. An "ABCDE mechanism" that can account for the ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.

* Corresponding author. Mailing address: Department of Medicine, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Los Angeles, CA 90095. Phone: (310) 824-5340. Fax: (310) 206-8766. E-mail: rlehrer{at}mednet.ucla.edu

{triangledown} Published ahead of print on 6 July 2009.

Editor: B. A. McCormick

Infection and Immunity, September 2009, p. 4028-4040, Vol. 77, No. 9
0019-9567/09/$08.00+0     doi:10.1128/IAI.00232-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Wei, G., de Leeuw, E., Pazgier, M., Yuan, W., Zou, G., Wang, J., Ericksen, B., Lu, W.-Y., Lehrer, R. I., Lu, W. (2009). Through the Looking Glass, Mechanistic Insights from Enantiomeric Human Defensins. J. Biol. Chem. 284: 29180-29192 [Abstract] [Full Text]  


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