Helix Induction in Antimicrobial Peptides by Alginate in Biofilms

Bacterial exopolysaccharides provide protection against phagocytosis, opsonization, and dehydration and act as a major structural component of the extracellular matrix in biofilms. They contribute to biofilm-related resistance by acting as a diffusion barrier to positively charged antimicrobial agents including cationic antimicrobial peptides (CAPs). We previously created novel CAPs consisting of a nonamphipathic hydrophobic core flanked by Lys residues and containing a Trp residue in the hydrophobic segment as a fluorescent probe. Peptides of this type above a specific hydrophobicity threshold insert spontaneously into membranes and have antimicrobial activity against Gram-positive and Gram-negative bacteria at micromolar concentrations. Here we show that alginate, a polymer of β-d-mannuronate and α-l-guluronate secreted by the cystic fibrosis pathogen Pseudomonas aeruginosa, induces an α-helical conformation detected by circular dichroism spectroscopy and blue shifts in Trp fluorescence maxima in peptides above the hydrophobicity threshold, changes typically observed upon association of such peptides with nonpolar (membrane) environments. Parallel effects were observed in the archetypical CAPs magainin II amide and cecropin P1. Fluorescence resonance energy transfer studies indicated that alginate induces peptide-peptide association only in peptides above the hydrophobicity threshold, suggesting that the hydrophilic alginate polymer behaves as an “auxiliary membrane” for the bacteria, demonstrating a unique protective role for biofilm matrices against CAPs. Bacterial exopolysaccharides provide protection against phagocytosis, opsonization, and dehydration and act as a major structural component of the extracellular matrix in biofilms. They contribute to biofilm-related resistance by acting as a diffusion barrier to positively charged antimicrobial agents including cationic antimicrobial peptides (CAPs). We previously created novel CAPs consisting of a nonamphipathic hydrophobic core flanked by Lys residues and containing a Trp residue in the hydrophobic segment as a fluorescent probe. Peptides of this type above a specific hydrophobicity threshold insert spontaneously into membranes and have antimicrobial activity against Gram-positive and Gram-negative bacteria at micromolar concentrations. Here we show that alginate, a polymer of β-d-mannuronate and α-l-guluronate secreted by the cystic fibrosis pathogen Pseudomonas aeruginosa, induces an α-helical conformation detected by circular dichroism spectroscopy and blue shifts in Trp fluorescence maxima in peptides above the hydrophobicity threshold, changes typically observed upon association of such peptides with nonpolar (membrane) environments. Parallel effects were observed in the archetypical CAPs magainin II amide and cecropin P1. Fluorescence resonance energy transfer studies indicated that alginate induces peptide-peptide association only in peptides above the hydrophobicity threshold, suggesting that the hydrophilic alginate polymer behaves as an “auxiliary membrane” for the bacteria, demonstrating a unique protective role for biofilm matrices against CAPs. Pseudomonas aeruginosa is the predominant respiratory tract pathogen in cystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; CAP, cationic antimicrobial peptide; FRET, fluorescence resonance energy transfer; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; TAMRA, carboxytetramethylrhodamine.1The abbreviations used are: CF, cystic fibrosis; CAP, cationic antimicrobial peptide; FRET, fluorescence resonance energy transfer; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; TAMRA, carboxytetramethylrhodamine. patients, where chronic infection is due to the bacteria growing as a mucoid biofilm, a state characterized by overproduction of alginate (1Lam J. Chan R. Lam K. Costerton J.W. Infect. Immun. 1980; 28: 546-556PubMed Google Scholar, 2Singh P.K. Schaefer A.L. Parsek M.R. Moninger T.O. Welsh M.J. Greenberg E.P. Nature. 2000; 407: 762-764Crossref PubMed Scopus (1198) Google Scholar, 3Costerton J.W. Stewart P.S. Greenberg E.P. Science. 1999; 284: 1318-1322Crossref PubMed Scopus (8769) Google Scholar). Alginate is a secreted extracellular polysaccharide composed of the uronic acid β-d-mannuronate and its C-5 epimer α-l-guluronate (4Gacesa P. Microbiology. 1998; 144: 1133-1143Crossref PubMed Scopus (143) Google Scholar), which is partially O-acetylated at the second and/or third position(s) of the d-mannuronate residues. The chronicity of P. aeruginosa infections in CF; its high level intrinsic antimicrobial resistance, a result of the low permeability of its outer membrane to antibiotics and multidrug efflux systems; and its propensity to develop resistance during prolonged antimicrobial therapy have all presented major therapeutic challenges to CF caregivers. Our laboratory has developed a new category of cationic antimicrobial peptides that display antibacterial activity (5Stark M. Liu L.P. Deber C.M. Antimicrob. Agents Chemother. 2002; 46: 3585-3590Crossref PubMed Scopus (175) Google Scholar). The peptides consist of a nonamphipathic hydrophobic core sequence (11-19 residues) flanked at one or both termini by a number of Lys or Arg residues. These peptides, which were originally designed as transmembrane mimetic model peptides (6Liu L.P. Li S.C. Goto N.K. Deber C.M. Biopolymers. 1996; 39: 465-470Crossref PubMed Scopus (68) Google Scholar, 7Liu L.P. Deber C.M. Biopolymers. 1998; 47: 41-62Crossref PubMed Scopus (102) Google Scholar), have the prototypic sequence KKAAAXAAAAAX- AAWAAXAAAKKKK-NH2 with several key features: (i) Ala is the preferred background residue because of its mid-range hydropathy and frequent occurrence in membrane protein transmembrane domains; (ii) a Trp residue is inserted into the hydrophobic segment as a fluorescent probe; and (iii) N- and C-terminal hydrophilic Lys residues act to solubilize the otherwise hydrophobic peptides in aqueous media to facilitate purification and characterization. These peptides have been shown to spontaneously insert into membranes when the average hydrophobicity of their core segment is above an experimentally determined “threshold” value based on the Liu-Deber hydrophobicity scale (see Table I), approximately above that where Xaa = Ala (7Liu L.P. Deber C.M. Biopolymers. 1998; 47: 41-62Crossref PubMed Scopus (102) Google Scholar). We have previously shown that peptides in this general category containing sequences above the hydrophobicity threshold have antimicrobial activity against both planktonic Gram-positive and Gram-negative bacteria at μg/ml concentrations (5Stark M. Liu L.P. Deber C.M. Antimicrob. Agents Chemother. 2002; 46: 3585-3590Crossref PubMed Scopus (175) Google Scholar).Table ICore segment hydrophobicity of transmembrane mimic peptides and alginate-induced changes in tryptophan fluorescence emission maxima (Δλmax)Peptide Xaa residueaPeptides have sequence KKAAAXAAAAAXAAWAAXAAAKKKK- NH2, where each contains three copies of a given Xaa residue. X indicates each of the 20 commonly occurring amino acids.Core segment hydrophobicitybAverage hydrophobicity of the core 19-residue segment based on Xaa residue relative hydrophobicity values on the Liu-Deber hydrophobicity scale (5). Peptides with average core hydrophobicity above 0.42 (X = Ala) are above the “threshold hydrophobicity” for membrane insertion (18).Alginate-induced blue shift (Δλmax)cThe blue shift is the shift in Trp emission maximum in a given peptide upon addition of alginate, given in nanometers as Δλmax = λmax (aqueous) - λmax (alginate).nmPhe1.1210Trp1.117Leu1.0912Ile1.0511Met0.868Val0.833Cys0.759Tyr0.689Ala0.425Thr, Glu, Asp, Gln, Arg, Ser, Gly, Asn, His, Pro, and Lys0.21 to −0.37All = 0a Peptides have sequence KKAAAXAAAAAXAAWAAXAAAKKKK- NH2, where each contains three copies of a given Xaa residue. X indicates each of the 20 commonly occurring amino acids.b Average hydrophobicity of the core 19-residue segment based on Xaa residue relative hydrophobicity values on the Liu-Deber hydrophobicity scale (5Stark M. Liu L.P. Deber C.M. Antimicrob. Agents Chemother. 2002; 46: 3585-3590Crossref PubMed Scopus (175) Google Scholar). Peptides with average core hydrophobicity above 0.42 (X = Ala) are above the “threshold hydrophobicity” for membrane insertion (18Deber C.M. Wang C. Liu L.P. Prior A.S. Agrawal S. Muskat B.L. Cuticchia A.J. Protein Sci. 2001; 10: 212-219Crossref PubMed Scopus (111) Google Scholar).c The blue shift is the shift in Trp emission maximum in a given peptide upon addition of alginate, given in nanometers as Δλmax = λmax (aqueous) - λmax (alginate). Open table in a new tab However, many antimicrobial compounds become considerably less effective against bacteria in biofilms. More specifically, alginate has been shown to act as a penetration barrier to conventional positively charged antibiotics through binding based on electrostatic interactions with the anionic sugars (8Nichols W.W. Dorrington S.M. Slack M.P.E. Walmsley H.L. Antimicrob. Agents Chemother. 1988; 32: 518-523Crossref PubMed Scopus (222) Google Scholar, 9Stewart P.S. Costerton J.W. Lancet. 2001; 358: 135-138Abstract Full Text Full Text PDF PubMed Scopus (3261) Google Scholar). However, the peptide-alginate interaction per se has not been widely studied. In this study, we investigated the effects of alginate on the structure of the 25-residue transmembrane mimic peptides. This set of model peptides allows us to vary the “guest” residue Xaa to any of the 20 commonly occurring amino acids individually and correlate the effects observed with the properties of the specific amino acid. By investigating the primary mode of interaction of these model peptides and the alginate polymers within biofilms, we can increase our understanding of the mechanism by which alginate may “trap” cationic antibiotics and ultimately progress toward optimizing the antimicrobial activity of this cationic antimicrobial peptide family against the P. aeruginosa biofilms in CF patients. Peptide Synthesis—The reagents for peptide synthesis, cleavage, and purification were Fmoc-protected amino acids (Novabiochem), [5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid]-polyethylene glycol-polystyrene (PAL-PEG-PS) resin (Applied Biosystems), N-N-dimethylformaamide (peptide grade; Caledon Laboratories Ltd.), piperidine (Applied Biosystems), methanol (Caledon), N,N-diisopropylethylamine (Aldrich), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (Applied Biosystems), diethyl ether (Caledon), triisopropylsilane (Aldrich), phenol (Invitrogen), and acetonitrile (Caledon). The peptides were synthesized by continuous flow Fmoc solid phase method on a PerSeptive Biosystems Pioneer peptide synthesizer using the standard cycle as previously described (7Liu L.P. Deber C.M. Biopolymers. 1998; 47: 41-62Crossref PubMed Scopus (102) Google Scholar). PAL-PEG-PS resin was used to produce an amidated C terminus. The O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate-N,N-diisopropylethylamine activator pair was used with amino acids at 4-fold excess. Deprotection and cleavage of the peptides were carried out in a mixture of 88% trifluoroacetic acid, 5% phenol, 5% water, and 2% triisopropylsilane for 2 h at room temperature. Cleaved and deprotected peptides were precipitated with ice-cold diethyl ether. Purification of peptides was carried out on a reversed-phase C4 preparative HPLC (21.2 × 250 mm, 300 A), using a linear gradient of acetonitrile in 0.1% trifluoroacetic acid as counter-ion. Purified peptides were characterized by analytical HPLC and mass spectrometry. Concentrations of peptides were determined in triplicate by micro-bicinchoninic acid protein assay. CD Spectroscopy—CD measurements were performed on a Jasco-720 spectropolarimeter using a 1-mm path-length quartz cell at 25 °C. Each spectrum was the average of four scans with buffer background or alginate in the absence of peptide subtracted. Peptide concentrations were typically 40-50 μm in 20 mm Tris-HCl (pH 7.0) in the presence and absence of carbohydrates. Seaweed alginate (Sigma) and alginate purified from P. aeruginosa (gift from Dr. Gerald Pier, Harvard Medical School) samples were extracted with both chloroform and hexane to remove potential fatty acid impurities (10Diefenbach R. Keweloh H. Rehm H.J. Appl. Microbiol. Biotechnol. 1992; 36: 530-534Crossref PubMed Scopus (22) Google Scholar) prior to use. Tryptophan Fluorescence Measurements—Fluorescence spectra were recorded on an Hitachi F-400 fluorescence spectrophotometer with emission and excitation slit widths of 4 nm. Fluorescence measurements were performed with 4 μm peptide solutions (20 mm Tris-HCl, pH 7.0) with or without the addition of 0.02 mg/ml carbohydrates at 25 °C with subtraction of the background of buffer or alginate in the absence of peptide. The excitation wavelength was 280 nm. The emission spectra were recorded from 300 to 400 nm. Fluorophore Labeling—Fluorophore labeling of peptides was performed as previously described (11Adair B.D. Engelman D.M. Biochemistry. 1994; 33: 5539-5544Crossref PubMed Scopus (137) Google Scholar). 5-Dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride), 4-dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl chloride), and 5-(and-6-)-carboxytetramethylrhodamine (5(6)-TAMRA), succinimidyl ester mixed isomers were obtained from Molecular Probes. Fluorescence Resonance Energy Transfer (FRET) Measurements—Separate populations of peptides were labeled with dansyl chloride as the donor chromophore and dabsyl chloride as the acceptor chromophore to observe association. The samples were prepared with 3 μm dansyl chloride-labeled peptide solutions (20 mm Tris-HCl, pH 7.0) with or without the addition of variable concentrations of dabsyl chloride-labeled peptide in the presence of 0.02 mg/ml alginate. Excitation wavelength was 341 nm. Emission spectra of the donor chromophore were recorded from 450 to 600 nm at 25 °C. Penetration of Peptides into Alginate Beads—Alginate beads (0.5 mm in diameter) were made by the “dripping method” (12Fundueanu G. Nastruzzi N. Carpov A. Desbrieres J. Rinaudo M. Biomaterials. 1999; 20: 1427-1435Crossref PubMed Scopus (259) Google Scholar) where droplets (1 μl) of a 1 mg/ml solution of alginate were added into a solution containing 1 m CaCl2. Following dialysis performed to remove excess CaCl2, 20 μl of 0.50 nm of TAMRA-labeled peptides were allowed to incubate with the alginate beads for 10 min. Unbound peptides were removed by washing with H2O. Examination of the interaction of TAMRA-labeled peptides with alginate beads was performed using a LSM 510 confocal laser scanning microscopy (Zeiss). The images were recorded with an excitation wavelength of 543 nm and an emission wavelength of 635 nm for TAMRA-labeled peptides at the mid-cross-section of the beads using the LSM 510 positioning software. Alginate Induces Helical Conformations in Peptides above the Hydrophobicity Threshold—In aqueous solution, the peptide with the guest residue Xaa = Phe displayed a partially helical CD spectrum (Fig. 1a) consistent with residue preferences on the Chou-Fasman (Pα) scale (7Liu L.P. Deber C.M. Biopolymers. 1998; 47: 41-62Crossref PubMed Scopus (102) Google Scholar, 13Chou P.Y. Fasman G.D. Biochemistry. 1974; 13: 211-222Crossref PubMed Scopus (1826) Google Scholar). However, upon the addition of alginate, the peptide adopted an essentially full α-helical conformation. Peptide interactions with alginate were seen to vary with peptide/alginate with CD spectra a of toward helical to the Xaa = Phe peptide (Fig. we that peptides above the hydrophobicity threshold of the Liu-Deber scale (7Liu L.P. Deber C.M. Biopolymers. 1998; 47: 41-62Crossref PubMed Scopus (102) Google Scholar) = and Ala) alginate α-helical in each of these peptides not In the spectra of peptides the threshold display of CD the spectra at or as an the model peptide with guest residue Xaa = is shown in peptides with guest residues and/or charged as Xaa = Arg, Asp, Glu, and displayed a variable for Xaa = in where alginate changes helical The that only peptides above the hydrophobicity threshold that the hydrophilic alginate polymer with peptides both electrostatic and hydrophobic interactions K. K. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Alginate Induces in Peptide Tryptophan Fluorescence Emission presence of Trp in the hydrophobic core of the model peptides allowed us to for changes in their by alginate-induced shifts occurring in the wavelength of their fluorescence emission The of fluorescence spectroscopy for interactions has been by M. Sci. S. A. PubMed Scopus Google Scholar). In the the emission maximum of each peptide was nm in aqueous solution, upon the addition of alginate, a blue shift in the emission for to a less by the increase in Table the blue shift values of the peptides in a alginate shifts of nm were observed for all peptides above the hydrophobicity threshold all the alginate on spectra of peptides the threshold with that alginate can from aqueous media PubMed Scopus Google Scholar), these the that alginate contains hydrophobic that may and blue effects in peptides above the hydrophobicity Molecular of on interactions that alginate insert into the by to insertion of membrane S. M. R. PubMed Scopus Google Scholar), an that alginate hydrophobic the alginate-induced blue shifts observed in fluorescence studies correlate with of in CD spectra of the set of peptides and hydrophobic peptide-alginate interactions are as a potential to the observed a for a hydrophobic in alginate, we the of the of its (Fig. a and α-l-guluronate of alginate in P. on its hydrophobic we that β-d-mannuronate contains a hydrophobic with a of four one in (Fig. of such alginate in a hydrophobic Fluorescence Resonance Energy Transfer of show that the anionic alginate can not only to cationic peptides through electrostatic and hydrophobic interactions can peptide-peptide we used to the interaction the of of to the termini of peptides. This has been widely used to interactions in protein J. Full Text PDF PubMed Google Scholar, J. 1992; PubMed Scopus Google Scholar), where and peptides in were used to that the protein in a (11Adair B.D. Engelman D.M. Biochemistry. 1994; 33: 5539-5544Crossref PubMed Scopus (137) Google Scholar). cationic antimicrobial peptides with guest residue Xaa = Phe were one labeled with dansyl chloride a and the labeled with dansyl chloride an shown in the emission maximum is when the Phe peptide was added to the peptide in alginate to with the was not a of increase in the of alginate upon the addition of Phe peptide not that the is not the result of peptides (11Adair B.D. Engelman D.M. Biochemistry. 1994; 33: 5539-5544Crossref PubMed Scopus (137) Google Scholar). to the observed energy transfer for a number of indicated that the peptide and/or in alginate solutions not of the interaction of the Phe peptide with alginate is the of a blue shift in the dansyl emission from nm in aqueous to nm in the presence of added alginate. In when the studies were performed with peptide with Xaa = Asp, of the dansyl chloride emission was observed when peptide was added in alginate solution (Fig. and blue shift in dansyl emission of by correlate the helical properties of the peptides in alginate to their to the alginate we investigated the penetration of the peptides in alginate Peptides with Xaa = Trp and Phe above the hydrophobicity and with Xaa = and the were labeled with with studies on labeled magainin S.C. 1998; PubMed Scopus Google Scholar), TAMRA-labeled peptides used in the displayed the antimicrobial activity against P. aeruginosa as their not seen in both the Trp and Phe peptides (Fig. a and were on the of the the and peptides (Fig. and into the of the CD of Peptides in the of the of alginate to α-helical in peptides with antimicrobial we performed on antimicrobial peptides. shown in the CD spectra in both cecropin A. M. H. S. A. PubMed Scopus Google Scholar) and M. S. A. 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