Cathelicidin peptides inhibit multiply antibiotic-resistant pathogens from patients with cystic fibrosis

Abstract

Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid P. aeruginosa strains (MIC, 0.06 to 8 microg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml). Peptides had modest activities against S. maltophilia and A. xylosoxidans (MIC range, 1.0 to > 32 microg/ml), but none inhibited B. cepacia. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P. aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.

FIG. 1

Time-kill studies. (A and B) P. aeruginosa PAO1 exposed to tobramycin (0.5 μg/ml), SMAP29 (2 μg/ml), or a combination of the two drugs. (A) Organisms after the first 8 h of exposure to each drug. (B) The organisms exposed to tobramycin and SMAP29 in combination were grown for 24 h and then rediluted and exposed to fresh tobramycin, SMAP29, or a combination of the two drugs (C) P. aeruginosa clinical strain 50BK exposed to tobramycin (1.0 μg/ml), SMAP29 (0.5 μg/ml), or SMAP29* (1.0 μg/ml).

FIG. 2

Activity of SMAP29 against P. aeruginosa in CF sputum. The activities against strains of P. aeruginosa in CF sputum of tobramycin (250 μg/ml), SMAP29 (250 or 1,000 μg/ml), and a PBS control with no antibiotics were compared. CFU/ml were determined after 10, 60, 240, 300, and 480 min of exposure to antibiotics as shown. The effects of the addition of a second dose at 240 min of tobramycin (final concentration, 500 μg/ml) or SMAP29 (final concentration, 500 or 2,000 μg/ml) are also shown. The arrow indicates the timing of the addition of the second dose of antibiotics. The results are representative of three replicate studies.

FIG. 3

Effects of SMAP29 or tobramycin treatment on the morphology of P. aeruginosa PAO1 evaluated by scanning electron microcopy. PAO1 was treated with media alone, tobramycin (5 μg/ml), or SMAP29 (0.5 μg/ml). At 1 min, 2 h, and 16 h the bacteria were processed for scanning electron microscopy. Within one minute, treatment with SMAP29 resulted in blebbing or blistering of the outer cell wall. The effects of tobramycin were much slower in their onset.

FIG. 4

Effects of cathelicidin peptides on the transepithelial resistance of primary cultures of human airway epithelia. Transepithelial resistance was measured at baseline and after 2.5 h (SMAP29, OV-1, and OV-2) or 4 h (LL37) of exposure to the peptides. The peptides were studied at concentrations of 1, 10, and 100 μg/ml. Transepithelial resistance values shown are normalized to 100% at zero hour. Following the peptide exposure measurement, the solution containing the peptide was removed and replaced with peptide-free solution for measurement of resistance at 24 h. Representative results are shown. The studies were performed three times in quadruplicate except for the LL37 studies, which were performed twice.