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Antimicrobial Agents and Chemotherapy, January 2001, p. 331-334, Vol. 45, No. 1
Respiratory Diseases of Livestock Research
Unit, USDA Agricultural Research Service, National Animal Disease
Center, Ames, Iowa 500101; Department of
Veterinary Pathology, Iowa State University, Ames, Iowa
500112; Midwest Area, USDA
Agricultural Research Service, Peoria, Illinois
616043; and Departments of
Pediatrics4 and
Microbiology,5 College of Medicine, The
University of Iowa, Iowa City, Iowa 52242
Received 14 February 2000/Returned for modification 8 May
2000/Accepted 2 October 2000
Cathelicidins are antimicrobial peptides from sheep (SMAP29 and
SMAP34), rabbits (CAP11 and CAP18), rodents (CRAMP), and humans (FALL39, LL37, and h/CAP18). In a broth microdilution assay against nine ovine pathogens, SMAP29, SMAP34, mouse CRAMP, CAP18,
CAP1831, CAP1828, CAP1822, and
CAP1821a were the most active, with MICs as low as 0.6 µg/ml. Other cathelicidins were less active. In lambs with pneumonia,
0.5 mg of SMAP29 reduced the concentration of bacteria in both
bronchoalveolar lavage fluid and consolidated pulmonary tissues. Hence,
the antimicrobial activity of SMAP29 suggests that it has applications
in the treatment of respiratory tract infections.
Innate host defenses in the
respiratory tract of sheep are currently being examined as models for
airway bacterial clearance (2), cystic fibrosis (2,
3, 10-13), and Pseudomonas aeruginosa pneumonia
(6, 7). SMAP29, a cathelicidin from sheep, is also being
considered as a therapeutic agent against microbial infections
(5, 9), including P. aeruginosa associated with chronic respiratory inflammation in cystic fibrosis patients
(14). Although SMAP29 is highly effective against P. aeruginosa PAO1 and other common gram-positive and gram-negative
organisms (14), its efficacy against a panel of pathogens
originating from the same host species is unknown. Therefore, a broth
microdilution assay was used to obtain both MICs and minimum
bactericidal concentrations (MBCs) of SMAP29 against a panel of ovine
pathogens (15, 18). SMAP34, mouse CRAMP, rat CRAMP,
FALL39, FF21, CAP18, and truncated, congeners of CAP18 were included
for comparison and synthesized as previously described
(14). Stock solutions of peptides were diluted in 0.4%
bovine serum albumin containing 0.02% acetic acid (0.16 to 80.00 µg/ml) and added to polypropylene microtiter plates (Sigma, St.
Louis, Mo.); 10 mM sodium phosphate buffer (pH 7.2) with 140 mM NaCl
(PBS) was added to control wells. Mueller-Hinton broth containing a
105-CFU/ml concentration of Mannheimia
haemolytica serovar 1 (strain 82-25), 2, or 6; Pasteurella
trehalosi serovar 4; Salmonella enterica subsp.
arizonae; ovine Pasteurella multocida serovar 3A
(strain P-2062); Klebsiella pneumoniae ATCC 10031;
Corynebacterium pseudotuberculosis ATCC 19410;
Staphylococcus aureus; or P. aeruginosa PAO1 was
added. Mueller-Hinton broth was added to wells containing PBS and used as the plate blank. After 24 and 48 h at 37°C, the optical
density of bacterial growth was determined (Spectromax Microplate
Reader; Molecular Devices Corp., Sunnyvale, Calif.). The MIC (e.g., the lowest concentration of peptide that reduced growth by more than 50%
compared to control wells) and the MBC (e.g., the lowest concentration of peptide that prevented well growth) were determined.
Peptides were active against PAO1, included as a susceptible control,
and these results (Tables 1 and
2) were similar to those previously
reported for other assays (14). Wide ranges of activity
against gram-negative bacteria were seen with mouse CRAMP (MIC, 3.3 to
15.0 µg/ml), rat CRAMP (MIC, 5.0 to 20.0 µg/ml), FALL39 (MIC, 2.1 to >20.0 µg/ml), FF21 (MIC, 3.3 to
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.331-334.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Ovine Cathelicidin SMAP29 Kills Ovine
Respiratory Pathogens In Vitro and in an Ovine Model of Pulmonary
Infection
ABSTRACT
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20.0 µg/ml), and SMAP34 (MIC,
5.0 to 11.7 µg/ml) (Table 1). MBCs were comparable to the
corresponding MICs. SMAP29 (MIC, 0.6 to 2.5 µg/ml) and CAP18 (MIC,
1.3 to 10.0 µg/ml) had the highest activities. The activity of CAP18
congeners also varied, and CAP1831, CAP1828, CAP1822, and CAP1821a were very active against
gram-negative bacteria, but the others were not (Table 2). All peptides
were less active against C. pseudotuberculosis and S. aureus (MIC, 10.0 to 20.0 µg/ml).
TABLE 1.
Antimicrobial activities of synthetic cathelicidins
against P. aeruginosa PAO1 and ovine pathogens
TABLE 2.
Antimicrobial activities of truncated congeners of CAP18
against P. aeruginosa PAO1 and ovine pathogens
In CAP18, truncations of the N terminus did not adversely affect the antimicrobial activity of congeners with a complete C terminus (e.g., CAP1831 and CAP1828), and truncations of the C terminus did not adversely affect the antimicrobial activity of congeners with a complete N terminus (e.g., CAP1821a). However, a severe truncation of the N terminus (e.g., CAP1826) or simultaneous truncations at both termini (e.g., CAP1821b, CAP1819, CAP1818, CAP1817, CAP1815a, and CAP1815b) decreased antimicrobial activity. Unfortunately, no central region with all of the antimicrobial activity could be identified. Instead, congeners needed to be at least 21 or more residues long to be effective. For PAO1, this number could drop to a minimum of 18 residues (e.g., CAP1818). Whether this size is related to the amphipathic structure of the peptide needed to form pores or otherwise disrupt the cytoplasmic membrane is not known.
The high activity of SMAP29 in these assays suggested that it might also be effective in vivo. To demonstrate this, 15 lambs, approximately 60 to 70 lb, were lightly sedated for bronchoscopy as previously described (1). Inocula were placed into the dorsum of the caudal portion of the cranial lobe of the right lung (pulmonary deposition site) in each animal. Two lambs in group 1 and three lambs in group 2 received 10 ml of PBS with 0.25 mg of purified ovine pulmonary surfactant per ml (PBS-PS). Five lambs in group 3 and five lambs in group 4 received 10 ml of M. haemolytica (2.5 × 108 CFU/ml in PBS-PS). PBS-PS was used as a diluent to decrease atelectasis and facilitate pulmonary spreading (4, 17). At 24 h postinoculation, lambs in group 1 and group 3 received an additional 10 ml of PBS-PS at the same deposition site. Lambs in group 2 received 10 ml of PBS-PS with 100 µg of SMAP/ml, and those in group 4 received 10 ml of PBS-PS with 50 µg of SMAP29/ml.
At 48 h postinoculation, all lambs were euthanized, their lungs were evaluated grossly, and total lung involvement was calculated as previously described (1, 16). Bronchoalveolar lavage (BAL) fluids and consolidated lung tissue were collected for quantitative bacteriological culture, determination of total leukocyte counts, and histopathological examination. The extents of lymphocytic and/or neutrophilic infiltration, necrosis, and collapse were scored as previously described (1), with a maximum score of 4 for each category. The concentrations of M. haemolytica in pulmonary tissues and fluids were transformed [log10 (X + 1)], and differences among groups was assessed by one-way analyses of variance. When an analysis of variance resulted in a significant F-test statistic, indicating group differences, Duncan's multiple-range test was used as the multiple comparison procedure for detecting pairwise differences among the treatment group means.
The lungs of lambs in groups 1 and 2 were free of gross lesions and
histopathology (Table 3). BAL fluids
contained means of 0.4 × 106 and 0.9 × 106 leukocytes/ml, respectively. Bronchioles and
surrounding alveoli contained minimal to mild infiltrates of
lymphocytes in the bronchiolar wall, but there was no acute
inflammatory response in the bronchioles or alveoli.
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The lungs of lambs in group 3 had extensive lesions characterized by focal areas of consolidation with hemorrhage and necrosis. BAL fluids contained a mean of 3.3 × 106 leukocytes/ml. There were minimal multifocal lymphocytic peribronchiolar infiltrates, mild to suppurative bronchitis and/or pneumonia, and minimal to moderate necrosis, hemorrhage, and collapse. Four of five lambs had M. haemolytica in BAL fluid (2.3 log10 CFU/ml), and all five lambs had organisms in consolidated pulmonary tissues (5.1 log10 CFU/g) collected from the deposition site. (Table 3).
The lesions in the lungs of lambs in group 4 were not as severe, and BAL fluids contained a mean of 4.9 × 106 leukocytes/ml. Sections of bronchioles and surrounding alveoli showed moderate numbers of lymphocytes in the bronchiolar wall but no acute inflammatory response. Three of five lambs had M. haemolytica in the BAL fluid (0.9 log10 CFU/ml), and all of the lambs had lesser numbers of organisms in consolidated pulmonary tissues (3.7 log10 CFU/g) collected from the deposition site.
Previously, prevention or treatment of respiratory infections with cathelicidins has not been fruitful. For example, CAP18 mixed with P. aeruginosa prior to intratracheal instillation in mice significantly reduced pulmonary injury but did not reduce the number of bacteria that had been instilled, nor did it improve the survival of the infected mice (8). Interestingly, CAP18 alone induced edema, suggesting that it had some pulmonary toxicity (8). To eliminate these species differences within the model, we established a homologous ovine model of acute pneumonia using ovine SMAP29 against the ovine respiratory pathogen M. haemolytica. SMAP29 alone was well tolerated (group 2), and the 1.0-mg dose (twice the treatment dose in group 4) did not induce any significant gross pathology, histopathology, or inflammatory cell filtrates in BAL fluid. Also, lambs treated with only a single dose of SMAP29 had substantially lower gross pulmonary lesion scores, histopathological lesion scores, and concentrations of M. haemolytica in BAL fluids and consolidated pulmonary tissues than untreated lambs. Further studies will determine the optimal doses and intervals of SMAP29 therapy.
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ACKNOWLEDGMENTS |
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We thank Gwen Laird and Abby Lozano for technical assistance.
This work was supported by the Cystic Fibrosis Foundation (BROGDE97Z, McCray 97ZO). P.B.M. is the recipient of a Career Development Award from the American Lung Association.
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FOOTNOTES |
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* Corresponding author. Mailing address: Respiratory Diseases of Livestock Research Unit, National Animal Disease Center, 2300 Dayton Ave., P.O. Box 70, Ames, IA 50010. Phone: (515) 663-7534. Fax: (515) 663-7458. E-mail: kbrogden{at}nadc.ars.usda.gov.
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Antimicrobial Agents and Chemotherapy, January 2001, p. 331-334, Vol. 45, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.331-334.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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