Bactericidal Activity of Mammalian Cathelicidin-Derived Peptides

S Travis

N Anderson

W Forsyth

C Espiritu

Departments of Microbiology,^{Internal Medicine,}^{Biochemistry,}^Pediatrics,^{and Physiology and Biophysics and Howard Hughes Medical Institute,}^{University of Iowa College of Medicine Iowa City, Iowa 52242, and UCLA Department of Medicine, Los Angeles, California 90095}³

^{Corresponding author. Mailing address: Department of Microbiology, Bowen Science Building, University of Iowa College of Medicine, Iowa City, IA 52242. Phone: (319) 335-8891. Fax: (319) 353-3038. E-mail:}ude.awoiu@kcat-nairb.

Received 1999 Nov 16; Revisions requested 1999 Dec 15; Accepted 2000 Jan 20.

Abstract

Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an α-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.

Abstract

The rapidly expanding prevalence of bacterial strains resistant to conventional antibiotics has prompted a search for new therapeutic agents, including various antimicrobial peptides of animal origin (15). Two broad classes of mammalian antibacterial peptides have been especially well studied: the cysteine-rich α- and β-defensins and various cathelicidins (6, 13, 22, 26, 27, 41, 42). Both classes are produced as precursors that require proteolytic processing to generate the mature antimicrobial peptide. Cathelicidins contain an N-terminal domain called cathelin, for which no function has yet been ascribed, and a C-terminal domain that comprises an antimicrobial peptide (reviewed in references 41 and 42). While the cathelin domains are highly conserved across species, the C-terminal antimicrobial domains are structurally diverse. The first cathelicidin precursor to be described was rabbit CAP18 (20), and its mature peptide was shown to have broad-spectrum bactericidal activity (19). Homologs of CAP18 have since been identified in other species including humans (FALL39/LL37) (1, 19), mice (mCRAMP) (12, 30), rats (rCRAMP), and sheep (SMAP29 and SMAP34) (2, 16, 25, 34). Circular dichroism (CD) measurements indicate that these linear peptides adopt α-helical structure in some solvents (1, 8, 12, 17, 36). These cathelicidin-derived peptides kill bacteria by disrupting the bacterial membrane (28).

Our primary goal in this study was to identify peptides of the cathelicidin family having intrinsically high bactericidal activity toward Pseudomonas aeruginosa and Staphylococcus aureus. These bacteria frequently manifest resistance to conventional antibiotics and pose serious problems for immunocompromised persons and cystic fibrosis patients. We also evaluated the physiochemical properties of each structure that correlated with antimicrobial activity to gain insights that could contribute to the rational design of salt-tolerant peptide antibiotics.

ACKNOWLEDGMENTS

We thank Elena Rus and Brian Morrison of the Protein Structure Facility at the University of Iowa for peptide synthesis, Matthew R. Parsek for construction of plasmid pMRP-77, and Michael A. Apicella for the lipopolysaccharide. We are grateful to Linda L. McCarter for her comments on the manuscript.

This work was supported by the National Institutes of Health (grants HL61234, AI29839, and AI43934), the Cystic Fibrosis Foundation, and the Howard Hughes Medical Institute.

ACKNOWLEDGMENTS

ADDENDUM IN PROOF

The peptide sequence of SMAP34 was based on the original description (K. M. Huttner, M. R. Lambeth, H. R. Burkin, D. J. Burkin, and T. E. Broad, Gene 206:85–91, 1998). The sequence has since been corrected (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"U60597","term_id":"1890244","term_text":"U60597"}}U60597).

ADDENDUM IN PROOF

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