Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

Respiratory Research

Neal L Schiller³  Robert I Lehrer¹  Olga Shamova²  Wei Wang²  Mark T Albrecht³ 

[1] Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA;Department of Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA;Division of Biomedical Sciences, University of California, Riverside, California 92521, USA

关键词: Pseudomonas aeruginosa;    protegrin;    lipopolysaccharide;    lipid A;    Burkholderia cepacia;   

Others  :  1227402
DOI  :  10.1186/rr167

 received in 2001-10-01, accepted in 2002-01-31,  发布年份 2002

【 摘 要 】

Background

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.

Methods

The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore).

Results

The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 μg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 μg/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains.

Conclusion

These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS.

【 授权许可】

   
2002 Albrecht et al, licencee BioMed Central Ltd

【 图 表 】

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【 参考文献 】

• [1]Drumm ML, Pope HA, Cliff WH, Rommens JM, Marvin SA, Tsui L-C, Collins FS, Frizzell RA, Willson JM: Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell 1990, 62:1227-1233.
• [2]Govan JRW, Deretic V: Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 1996, 60:539-574.
• [3]Hutchison ML, Govan JRW: Pathogenicity of microbes associated with cystic fibrosis. Microbes Infect 1999, 1:1005-1014.
• [4]Wine JJ: The genesis of cystic fibrosis lung disease. J Clin Invest 1999, 103:309-312.
• [5]Gilljam H, Ellin A, Strandvik B: Increased bronchial chloride concentration in cystic fibrosis. Scand J Lab Invest 1989, 49:121-124.
• [6]Joris L, Dab I, Quinton PM: Elemental composition of human airway surface fluid in healthy and diseased airways. Am Rev Respir Dis 1993, 148:1633-1637.
• [7]Comolli JC, Waite LL, Mostov KE, Engel JN: Pili binding to asialo-GM1 on epithelial cells can mediate cytotoxicity or bacterial internalization by Pseudomonas aeruginosa. Infect Immun 1999, 67:3207-3214.
• [8]Saiman L, Prince A: Pseudomonas aeruginosa pili bind to asialo-GM1 which is increased on the surface of cystic fibrosis epithelial cells. J Clin Invest 1993, 92:1875-1880.
• [9]Pier GB, Grout M, Zaidi TS, Olsen JC, Johnson LG, Yankaskas JR, Goldberg JB: Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections. Science 1996, 271:64-67.
• [10]Pier GB, Grout M, Zaidi TS: Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung. Proc Natl Acad Sci USA 1997, 94:12088-12093.
• [11]Pier GB: Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections. Proc Natl Acad Sci USA 2000, 97:8822-8828.
• [12]Bals R, Wang X, Wu Z, Freeman T, Bafna V, Zasloff M, Wilson JM: Human β-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung. J Clin Invest 1998, 102:874-880.
• [13]Goldman MJ, Anderson M, Stolzenberg ED, Kari UP, Zaasloff M, Wilson JM: Human β-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997, 88:553-560.
• [14]Lehrer RI, Lichtenstein AK, Ganz T: Defensins: Antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol 1993, 11:105-128.
• [15]Wang J, Lory S, Ramphal R, Jin S: Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients. Mol Microbiol 1996, 22:1005-1012.
• [16]Jayaraman S, Joo NS, Reitz B, Wine JJ, Verkman AS: Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na+] and pH but elevated viscosity. Proc Natl Acad Sci USA 2001, 98:8119-8123.
• [17]Johnston RB: Clinical aspects of chronic granulomatous disease. Curr Opin Hematol 2001, 8:17-22.
• [18]Speert DP: Understanding Burkholderia cepacia : epidemiology, genomovars, and virulence. Infect Med 2001, 18:49-56.
• [19]Hancock REW, Chappel DS: Peptide Antibiotics. Antimicrob Agents Chemother 1999, 43:1317-1323.
• [20]Kokryakov VN, Harwig SSL, Panyutich EA, Shevchenko AA, Alashina GM, Shamova OV, Korneva HA, Lehrer RI: Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins. FEBS Lett 1993, 327:231-236.
• [21]Harwig SSL, Waring AJ, Yang HJ, Cho Y, Tan L, Lehrer RI: Intramolecular disulfide bonds enhance the antimicrobial and lytic activities of protegrins at physiological sodium chloride concentrations. Eur J Biochem 1996, 240:352-357.
• [22]Steinberg DA, Hurst MA, Fujii CA, Kung AHC, Ho JF, Cheng F-C, Loury DJ, Fiddes JC: Protegrin-1: a broad-spectrum, rapidly microbicidal peptide with in vivo activity. Antimicrob Agents Chemother 1997, 41:1738-1742.
• [23]Hancock REW, Lehrer RI: Cationic peptides a new source of antibiotics. Trends Biotech 1998, 16:82-88.
• [24]Yasin B, Lehrer RI, Harwig SSL, Wagar EA: Protegrins: structural requirements for the inactivation of elementary bodies of Chlamydia trachomatis. Infect Immun 1996, 64:4863-4866.
• [25]Qu X-D, Harwig SSL, Shafer WM, Lehrer RI: Protegrin structure and activity against Neisseria gonorrhoea. Infect Immun 1997, 65:636-639.
• [26]Mangoni ME, Aumelas A, Charnet P, Roumestand C, Chiche L, Despaux E, Grassy G, Calas B, Chavanieu A: Change in membrane permeability induced by protegrin-1: implication of disulphide bridges for pore formation. FEBS Lett 1996, 383:93-98.
• [27]Aumelas A, Magoni M, Roumestand C, Chiche L, Despaux E, Grassy G, Calas B, Chavanieu A: Synthesis and solution structure of the antimicrobial peptide protegrin-1. Eur J Biochem 1996, 237:575-583.
• [28]Fahrner RL, Dieckmann T, Harwig SSL, Lehrer RI, Eisenberg D, Feigon J: Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes. Chemistry Biol 1996, 3:543-550.
• [29]Cho Y, Turner JS, Dinh N-N, Lehrer RI: Activity of protegrins against yeast-phase Candida albicans. Infect Immun 1998, 66:2486-2493.
• [30]Fortney K, Totten PA, Lehrer RI, Spinola SM: Haemophilus ducreyi is susceptible to protegrin. Antimicro Agents Chemother 1998, 42:2690-2693.
• [31]Miyasaki KT, Lofel R, Oren A, Huynh T, Lehrer RI: Killing of Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia by protegrins. J Periodont Res 1998, 33:91-98.
• [32]Yasin B, Harwig SSL, Lehrer RI, Wagar EA: Susceptibility of Chlamydia trachomatis to protegrins and defensins. Infect Immun 1996, 64:709-713.
• [33]Schiller NL, Hackley DR, Morrison A: Isolation and characterization of serum-resistant strains of Pseudomonas aeruginosa derived from serum-sensitive parental strains. Curr Microbiol 1984, 10:185-190.
• [34]Ohman DE, Chakrabarty AM: Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun 1981, 33:142-148.
• [35]Ohman DE, Chakrabarty AM: Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin. Infect Immun 1982, 37:662-669.
• [36]LiPuma JJ, Spilker T, Gill LH, Campbell PW III, Liu L, Mahenthiralingam E: Disproportionate distribution of Burkholderia cepacia complex species and transmissibility markers in cystic fibrosis. Am J Respir Crit Care Med 2001, 164:92-96.
• [37]Steinberg DA, Lehrer RI: Designer assays for antimicrobial peptides: disputing the "one size fits all" theory. Methods in Mol Biol 1997, 78:169-186.
• [38]Lehrer RI, Rosenman M, Harwig SSL, Jackson R, Eisenhauer P: Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods 1991, 137:167-173.
• [39]Hultmark D, Engstrom A, Andersson K, Steiner H, Bennich H, Boman GH: Insect immunity, attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO 1983, 2:571-576.
• [40]Hultmark D, Engstrom A, Hennich H, Kapur R, Boman GH: Isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. Eur J Biochem 1982, 127:207-217.
• [41]Lehrer RI, Szklarek D, Ganz T, Selsted M: Correlation of the binding of rabbit granulocyte peptides to Candida albicans with candidacidal activity. Infect Immun 1985, 49:207-211.
• [42]Tsomides T, Eisen H: Stoichiometric labeling of peptides by iodination of tyrosyl or histidyl residues. Anal Biochem 1993, 210:129-135.
• [43]Darveau RP, Hancock REW: Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol 1983, 155:831-838.
• [44]Mattsby-Baltzer I, Kaijser B: Lipid A and anti-lipid A. Infect Immun 1979, 23:758-763.
• [45]Qureshi N, Takayama K: Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium. J Biol Chem 1982, 257:11808-11815.
• [46]Tan NS, Ng ML, Yau YH, Chong PK, Ho B, Ding JL: Definition of endotoxin binding sites in horseshoe crab factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides. FASEB J. 2000, 14:1801-1813.
• [47]Heller WT, Waring AJ, Lehrer RI, Harroun TA, Weiss TM, Yang L, Huang HW: Membrane thinning effect of the β-sheet antimicrobial protegrin. Biochem 2000, 39:139-145.
• [48]Freer E, Moreno E, Moriyon I, Pizarro-Cerda J, Weintraub A, Gorvel J-P: Brucella-salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts. J Bacteriol 1996, 178:5867-5876.
• [49]Hancock REW, Bell A: Antibiotic uptake into Gram-negative bacteria. Eur J Clin Microbiol Infect Dis 1988, 7:713-720.
• [50]Martinez de Tejada G, Pizarro-Cerada J, Moreno E, Moriyon I: The outer membranes of Brucella spp. are resistant to bactericidal cationic peptides. Infect Immun 1995, 63:3054-3061.
• [51]Piers KL, Brown MH, Hancock REW: Improvement of outer membrane-permeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification. Antimicrob Agents Chemother 1994, 38:2311-2316.
• [52]Gunn JS, Ryan SS, Van Velkinburgh JC, Ernest RK, Miller SI: Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium. Infect Immun 2000, 68:6139-6146.
• [53]Gunn JS, Lim KB, Krueger J, Kim K, Guo L, Hackett M, Miller SI: PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol Microbiol 1998, 27:1171-1182.
• [54]Moore RA, Hancock REW: Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance. Antimicrob Agents Chemother 1986, 30:923-926.
• [55]Skurnik M, Venho R, Bengoechea JA, Moriyon I: The lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3 is required for virulence and plays a role in outer membrane integrity. Mol Microbiol 1999, 31:1443-1462.
• [56]Bengoechea JA, Skurnik M: Temperature-regulated efflux pump/potassium antiporter system mediates resistance to cationic antimicrobial peptides in Yersinia. Mol Microbiol 2000, 37:67-80.
• [57]Shafer WM, Qu X, Waring AJ, Lehrer RI: Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/modulation/division efflux pump family. Proc Natl Acad Sci U S A 1998, 95:1829-1833.
• [58]Cox AD, Wilkinson SG: Ionizing groups in lipopolysaccharides of Pseudomonas cepacia in relation to antibiotic resistance. Mol Microbiol 1991, 5:641-646.
• [59]Gunn JS, McCoy AJ, Tran L, Liu H, Falla T: Identification and characterization of Burkholderia cepacia mutants sensitive to antimicrobial peptides, abstr A92, p. 22. In Abstracts of the American Society for Microbiology: 101st General Meeting 2001, abstr A92:22.
• [60]Manniello JM, Heymann H, Adair FW: Isolation of atypical lipopolysaccharides from purified cell walls of Pseudomonas cepacia. J Gen Microbiol 1979, 29:496-500.
• [61]Ernst RK, Yi EC, Guo L, Lim KB, Burns JL, Hackett M, Miller SI: Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science 1999, 286:1561-1565.
• [62]Wimmera N, Bradeb H, Kosma P: Synthesis of neoglycoproteins containing D-glycero-D-talo-oct-2-ulopyranosylonic acid (KO) ligands corresponding to core units from Burkholderia and Acinetobacter lipopolysaccharide. Carb Res 2000, 329:549-560.