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Infection and Immunity, November 2009, p. 5035-5043, Vol. 77, No. 11
0019-9567/09/$08.00+0     doi:10.1128/IAI.00695-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Electropositive Charge in -Defensin Bactericidal Activity: Functional Effects of Lys-for-Arg Substitutions Vary with the Peptide Primary Structure ^,

R. Alan Llenado,^1, Colby S. Weeks,^1,, Melanie J. Cocco,² and André J. Ouellette^1,3*

Departments of Pathology & Laboratory Medicine,¹ Molecular Biology & Biochemistry,² Microbiology & Molecular Genetics, Schools of Medicine and Biological Sciences, University of California, Irvine, California 92697-4800³

Received 18 June 2009/ Returned for modification 7 July 2009/ Accepted 30 August 2009

Cationic amino acids contribute to -defensin bactericidal activity. Curiously, although Arg and Lys have equivalent electropositive charges at neutral pH, -defensins contain an average of nine Arg residues per Lys residue. To investigate the role of high -defensin Arg content, all Arg residues in mouse Paneth cell -defensin cryptdin 4 (Crp4) and rhesus myeloid -defensin 4 (RMAD-4) were replaced with Lys to prepare (R/K)-Crp4 and (R/K)-RMAD-4, respectively. Lys-for-Arg replacements in Crp4 attenuated bactericidal activity and slowed the kinetics of Escherichia coli ML35 cell permeabilization, and (R/K)-Crp4 required longer exposure times to reduce E. coli cell survival. In marked contrast, Lys substitutions in RMAD-4 improved microbicidal activity against certain bacteria and permeabilized E. coli more effectively. Therefore, ArgLys substitutions attenuated activity in Crp4 but not in RMAD-4, and the functional consequences of ArgLys replacements in -defensins are dependent on the peptide primary structure. In addition, the bactericidal effects of (R/K)-Crp4 and (R/K)-RMAD-4 were more sensitive to inhibition by NaCl than those of the native peptides, suggesting that the high Arg content of -defensins may be under selection to confer superior microbicidal function under physiologic conditions.

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* Corresponding author. Mailing address: Department of Pathology & Laboratory Medicine, Keck School of Medicine of The University of Southern California, USC Norris Cancer Center, 1450 Biggy Street, NRT 7514 Mail Code 9601, Los Angeles, CA 90033. Phone: (323) 442-7959. Fax: (323) 442-7962. E-mail: aouellet{at}usc.edu

Published ahead of print on 8 September 2009.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: F. C. Fang

Contributed equally to these studies.

Present address: Department of Medical Microbiology and Immunology, University of California, Davis, Davis, CA 95616.

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Infection and Immunity, November 2009, p. 5035-5043, Vol. 77, No. 11
0019-9567/09/$08.00+0     doi:10.1128/IAI.00695-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.