Structure and Mechanism of Action of an Indolicidin Peptide Derivative with Improved Activity against Gram-positive Bacteria
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m009691200
ISSN1083-351X
AutoresCarol L. Friedrich, Annett Rozek, Aleksander Patrzykat, Robert E. W. Hancock,
Tópico(s)Immune Response and Inflammation
ResumoIndolicidin, an antimicrobial peptide with a unique amino acid sequence (ILPWKWPWWPWRR-NH2) is found in bovine neutrophils. A derivative of indolicidin, CP10A, has alanine residues substituted for proline residues and has improved activity against Gram-positive organisms. Transmission electron microscopy of Staphylococcus aureus andStaphylococcus epidermidis treated with CP10A showed mesosome-like structures in the cytoplasm. The peptide at 2-fold the minimal inhibitory concentration did not show significant killing ofS. aureus ISP67 (a histidine, uridine, and thymidine auxotroph) but did show an early effect on histidine and uridine incorporation and, later, an effect on thymidine incorporation. Upon interaction with liposomes, detergents, and lipoteichoic acid, CP10A was shown by circular dichroism spectroscopy to undergo a change in secondary structure. Fluorescence spectroscopy indicated that the tryptophan residues were located at the hydrophobic/hydrophilic interface of liposomes and detergent micelles and were inaccessible to the aqueous quencher KI. The three-dimensional structure of CP10A in the lipid mimetic dodecylphosphocholine was determined using two-dimensional NMR methods and was characterized as a short, amphipathic helical structure, whereas indolicidin was previously shown to have an extended structure. These studies have introduced a cationic peptide with a unique structure and an ability to interact with membranes and to affect intracellular synthesis of proteins, RNA, and DNA. Indolicidin, an antimicrobial peptide with a unique amino acid sequence (ILPWKWPWWPWRR-NH2) is found in bovine neutrophils. A derivative of indolicidin, CP10A, has alanine residues substituted for proline residues and has improved activity against Gram-positive organisms. Transmission electron microscopy of Staphylococcus aureus andStaphylococcus epidermidis treated with CP10A showed mesosome-like structures in the cytoplasm. The peptide at 2-fold the minimal inhibitory concentration did not show significant killing ofS. aureus ISP67 (a histidine, uridine, and thymidine auxotroph) but did show an early effect on histidine and uridine incorporation and, later, an effect on thymidine incorporation. Upon interaction with liposomes, detergents, and lipoteichoic acid, CP10A was shown by circular dichroism spectroscopy to undergo a change in secondary structure. Fluorescence spectroscopy indicated that the tryptophan residues were located at the hydrophobic/hydrophilic interface of liposomes and detergent micelles and were inaccessible to the aqueous quencher KI. The three-dimensional structure of CP10A in the lipid mimetic dodecylphosphocholine was determined using two-dimensional NMR methods and was characterized as a short, amphipathic helical structure, whereas indolicidin was previously shown to have an extended structure. These studies have introduced a cationic peptide with a unique structure and an ability to interact with membranes and to affect intracellular synthesis of proteins, RNA, and DNA. dodecylphosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1,2-dioleoyl-sn-glycero-3-phosphotempocholine 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine 1-palmitoyl-2-stearoyl-(12-doxyl)-sn-glycero-3-phosphocholine minimal inhibitory concentration circular dichroism total correlation spectroscopy nuclear Overhauser effect (/enhancement) spectroscopy double quantum-filtered correlation spectroscopy Antimicrobial cationic peptides are ubiquitous in nature and are thought to be an important component in innate host defenses against infectious agents (1Hancock R.E.W. Falla T.J. Brown M. Adv. Microb. Physiol. 1995; 37: 136-175Google Scholar). There are four structural classes of cationic antimicrobial peptides; the disulfide-bonded β-sheet peptides, including the defensins; the amphipathic α-helical peptides such as the cecropins and melittins; the extended peptides, which often have a single amino acid predominating (e.g. indolicidin); and the loop-structured peptides like bactenecin (1Hancock R.E.W. Falla T.J. Brown M. Adv. Microb. Physiol. 1995; 37: 136-175Google Scholar). The initial interactions of some cationic peptides with Gram-negative bacteria are thought to involve binding to surface lipopolysaccharide (2Piers K.L. Hancock R.E.W. Mol. Microbiol. 1994; 12: 951-958Crossref PubMed Scopus (78) Google Scholar, 3Sawyer J.G. Martin N.L. Hancock R.E. Infect. Immun. 1988; 56: 693-698Crossref PubMed Google Scholar). The peptides displace divalent cations that are essential for outer membrane integrity and consequently distort the outer membrane bilayer (4Peterson A.A. Fesik S.W. McGroarty E.J. Antimicrob. Agents Chemother. 1987; 31: 230-237Crossref PubMed Scopus (58) Google Scholar). This allows access to the cytoplasmic membrane where peptide channel formation has been proposed to occur (5Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (584) Google Scholar). It is increasingly disputed as to whether peptide channel formation leads to dissolution of the proton motive force and leakage of essential molecules (6Cociancich S. Ghazi A. Hoffman J.A. Hetrus C. Letellier C. J. Biol. Chem. 1993; 268: 19239-19245Abstract Full Text PDF PubMed Google Scholar, 7Juretic D. Chan H.C. Brown J.H. Morell J.L. Hendler R.W. Westerhoff H. FEBS Lett. 1989; 249: 219-223Crossref PubMed Scopus (76) Google Scholar) or whether it is an intermediate step in the uptake of peptide into the cytoplasm, where it inhibits an essential function by e.g.binding to polyanionic DNA (8Wu M. Maier E. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 7235-7242Crossref PubMed Scopus (631) Google Scholar, 9Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (694) Google Scholar, 10Zhang L. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 8102-8111Crossref PubMed Scopus (134) Google Scholar). Indolicidin is a 13-amino acid cationic peptide present in the cytoplasmic granules of bovine neutrophils (11Selsted M.E. Novotny M.J. Morris W.L. Tang Y.Q. Smith W. Cullor J.S. J. Biol. Chem. 1992; 267: 4292-4295Abstract Full Text PDF PubMed Google Scholar). Indolicidin has a unique amino acid composition (ILPWKWPWWPWRR-NH2) with 39% tryptophan, 23% proline, and an amidated carboxyl terminus. This, along with the fact that it has a broad spectrum of antimicrobial activity, has made it an interesting candidate for study. The structure of indolicidin was determined by two-dimensional NMR and shown to form an extended boat-shaped conformation when bound to dodecylphosphocholine (DPC)1(12Rozek A. Friedrich C.L. Hancock R.E.W. Biochemistry. 2000; 39: 15765-15774Crossref PubMed Scopus (276) Google Scholar). Previous structure-function studies by Subbalakshmi et al. (13Subbalakshmi C. Krishnakumari V. Nagaraj R. Sitaram N. FEBS Lett. 1996; 395: 48-52Crossref PubMed Scopus (120) Google Scholar) led to the design of a peptide ILA, called here CP10A. CP10A is a peptide with the three proline residues of indolicidin replaced with alanine, resulting in the amino acid sequence ILAWKWAWWAWRR-NH2. Subbalakshmi et al. (13Subbalakshmi C. Krishnakumari V. Nagaraj R. Sitaram N. FEBS Lett. 1996; 395: 48-52Crossref PubMed Scopus (120) Google Scholar) found that these amino acid substitutions had no effect on the activity against Escherichia coli and Staphylococcus aureus (13Subbalakshmi C. Krishnakumari V. Nagaraj R. Sitaram N. FEBS Lett. 1996; 395: 48-52Crossref PubMed Scopus (120) Google Scholar). However, our previous studies show that CP10A has 2–8-fold better activity against most Gram-positive bacteria (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). In the present work we examined the structure and the mode of action of CP10A against Gram-positive bacteria in greater detail using a variety of biophysical and biochemical methods. Various studies of the effects of cationic peptides on the membranes of Gram-positive bacteria have been conducted. Previous studies of peptide effects on membrane potential show that there are effects on the cytoplasmic membrane of S. aureus (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar, 15Yeaman M.R. Bayer A.S. Koo S.P. Foss W. Sullam P.M. J. Clin. Invest. 1998; 101: 178-187Crossref PubMed Scopus (165) Google Scholar). As well, ultrastructural studies of S. aureus treated with defensins (16Shimoda M. Ohki K. Shimamoto Y. Kohashi O. Infect. Immun. 1995; 63: 2886-2891Crossref PubMed Google Scholar) and platelet microbicidal proteins (15Yeaman M.R. Bayer A.S. Koo S.P. Foss W. Sullam P.M. J. Clin. Invest. 1998; 101: 178-187Crossref PubMed Scopus (165) Google Scholar) showed cell membrane damage followed by cell death. The defensins caused mesosome-like structures to appear before the bacteria lost their viability, but no remarkable effects on the cell wall were seen (16Shimoda M. Ohki K. Shimamoto Y. Kohashi O. Infect. Immun. 1995; 63: 2886-2891Crossref PubMed Google Scholar). These studies suggested that membrane perturbation is an important, but not necessarily lethal, event. Recent studies indicate that some peptides may indeed have an intracellular target. Xiong et al. (17Xiong Y.Q. Yeaman M.R. Bayer A.S. Antimicrob. Agents Chemother. 1999; 43: 1111-1117Crossref PubMed Google Scholar) find that S. aureus, pretreated with inhibitors of DNA gyrase or protein synthesis, demonstrated decreased or blocked killing by human neutrophil peptide-1 and platelet microbicidal protein-1, whereas pretreatment with bacterial cell wall synthesis inhibitors enhanced bacterial killing. The authors concluded that these cytoplasmic membrane effects occurred before effects on protein and DNA synthesis. As well, in our lab we demonstrated a lack of correlation between bacterial killing and cytoplasmic membrane depolarization and, in Staphylococcus epidermidis, nuclear condensation, indicating effects on DNA (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). There is thus growing evidence for an intracellular target for some antimicrobial cationic peptides. Here we describe a peptide with a unique structure that causes ultrastructural effects on S. aureus and S. epidermidis similar to those seen with other cationic peptides as well as intracellular effects. CP10A (ILAWKWAWWAWRR-NH2) was synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry at the Nucleic Acid Protein Service (NAPS) unit at the University of British Columbia. The peptide was pure, as confirmed by high performance liquid chromatography and mass spectrometry. Bovine serum albumin fraction V lyophilizate was purchased from Roche Molecular Biochemicals. S. aureus ATCC 25923, and S. epidermidis (clinical isolate, obtained from David Speert, University of British Columbia) (18Scott M.G. Gold M.R. Hancock R.E.W. Infect. Immun. 1999; 67: 6445-6453Crossref PubMed Google Scholar) were used for electron microscopy. S. aureus ISP67 (obtained from John Iandolo, Kansas State University), a strain auxotrophic for thymidine, uridine, and histidine, was used in the macromolecular synthesis studies. In most cases Luria-Bertani media (no salt) (Difco) was used as a growth medium, with the exception ofS. aureus ISP67, which was grown in modified complete synthetic media (salts, glucose, amino acids, Lindberg vitamins, niacin, and thiamine) supplemented with 20 mg/liter thymidine, 5 mg/liter uridine, and 20 mg/liter histidine (Sigma). Dipropylthiacarbocyanine (5Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (584) Google Scholar) was purchased from Molecular Probes (Eugene, OR). Perdeuterated DPC (DPC-d38) and deuterium oxide (D2O) were purchased from Cambridge Isotope Laboratories, Andover, MA. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were purchased from Northern Lipids Inc., Vancouver, BC, Canada. 1,2-Dioleoyl-sn-glycero-3-phosphotempocholine (head group labeled tempo-PC), 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine (5-doxyl-PC), and 1-palmitoyl-2-stearoyl-(12-doxyl)-sn-glycero-3-phosphocholine (12-doxyl-PC) were obtained from Avanti Polar Lipids Inc., Alabaster, AL. Exponential phase bacteria were treated with the peptide at 10 times the minimal inhibitory concentration (MIC) for 10 min at 37 °C. This concentration was used to see an effect on a greater fraction of cells. After treatment, the bacterial pellets were fixed with 2.5% buffered glutaraldehyde for 1 h. The cells were then post-fixed in 1% buffered osmium tetroxide for 1 h, stained en bloc with 1% uranyl acetate, dehydrated in a graded series of ethanol, and embedded in Spur resin. The buffer used was 0.1 m sodium cacodylate (pH 7.4). Thin sections were prepared on Formvar copper grids and stained with 2% uranyl acetate and lead citrate. The resin and grids were purchased from Canemco (Toronto, ON, Canada). Microscopy was performed with a Zeiss Stem 10C microscope under standard operating conditions. The MIC of CP10A was determined using a broth microdilution assay modified from the method of Amsterdam (19Amsterdam D. Lorian V. Antibiotics in Laboratory Medicine. 4th Ed. Lippincott Williams & Wilkins, Baltimore1996: 52-111Google Scholar). Briefly, serial dilutions of the peptide were made in 0.2% bovine serum albumin, 0.01% acetic acid solution in 96-well polypropylene (Costar, Corning Inc., Corning, NY) microtiter plates. Each well was inoculated with 100 μl of the test organism in Luria-Bertani (no salt) broth or in the synthetic media to a final concentration of ∼105 colony-forming units/ml. The MIC was taken as the lowest peptide concentration at which growth was inhibited after 24 h of incubation at 37 °C. The depolarization of the cytoplasmic membrane of S. aureus by the peptides was determined using the membrane potential-sensitive cyanine dye dipropylthiacarbocyanine (5Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (584) Google Scholar) (20Sims P.J. Waggoner A.S. Wang C.H. Hoffman J.F. Biochemistry. 1974; 13: 3315-3330Crossref PubMed Scopus (764) Google Scholar) by a modification of the method of Wu et al. (8Wu M. Maier E. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 7235-7242Crossref PubMed Scopus (631) Google Scholar), as described in Friedrich et al. (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). Overnight cultures of S. aureus ISP67 were diluted 100-fold in synthetic media and allowed to grow to exponential phase (optical density at 600 nm of 0.3). The cultures were spun down and resuspended in warm synthetic media with 20 mg/liter [3H]thymidine, 5 mg/liter [3H]uridine, or 20 mg/liter [3H]histidine. After 5 min of incubation at 37 °C, CP10A was added at 2- and 10-fold its MIC. Samples (50 μl) were removed at 0 min (before peptide) and 5, 10, 20, and 40 min and added to cold 5% trichloroacetic acid (purchased from Fisher Scientific) with excess unlabeled precursors to precipitate the macromolecules. After 40 min on ice and 15 min at 37 °C, the samples were collected over a vacuum on Whatman 47 mm GF/C glass microfibre filters (VWR Canlab, Mississauga, ON, Canada) and washed with cold trichloroacetic acid. The filters were collected and put into scintillation vials with ReadySafe liquid scintillation mixture (Beckman Instruments) and counted on a scintillation counter to measure precursor incorporation into macromolecules. At the same time points, 5-μl samples were removed from nonradioactive parallel cultures, diluted in 1 ml of buffer, and plated on to LB plates with added supplements to obtain a viable count. A chloroform solution of lipid was mixed with the peptide dissolved in methanol. This solution was dried under a stream of N2 in vacuum to remove the solvent. The resulting lipid/peptide film was rehydrated in 10 mmphosphate buffer (pH 7.0). The suspension was put through five cycles of freeze-thaw to produce multilamellar liposomes, followed by extrusion through a 0.1-μm double-stacked Poretics filters (AMD Manufacturing Inc., Mississauga, ON, Canada) using an extruder device (Lipex Biomembranes, Vancouver, BC, Canada). Fluorescence emission spectra were recorded on an LS 50B spectrofluorimeter (PerkinElmer Life Sciences). Measurements were performed between 300 and 450 nm at 1-nm increments using a 5-mm quartz cell at room temperature. The excitation wavelength was set to 280 nm with both the excitation and emission slit widths set to 4 nm. Spectra were base-line-corrected by subtracting blank spectra of the corresponding lipid or detergent solutions without peptide. The samples contained 2 μmpeptide and 0.5 mm lipid or 10 mm detergent in 10 mm HEPES buffer (pH 7.2). The aqueous quencher potassium iodide was added in increasing increments to assess tryptophan accessibility to the aqueous buffer. Spin labeled lipids were used to estimate the tryptophan position in the liposomes. Circular Dichroism (CD) spectra were obtained using a J-720 spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan). Each spectrum (190–250 nm) was the average of 4 scans using a quartz cell of 1-mm path length at room temperature. The scanning speed was 50 nm/min at a step size of 0.1 nm, 2 s response time and 1.0-nm bandwidth. All samples were 50 μm peptides in 10 mm sodium phosphate buffer (pH 7.0). The concentrations of lipid or detergent were 2 or 10 mm, respectively. Spectra were base-line-corrected by subtracting a blank spectrum of a sample containing all components except the peptide. After noise correction, ellipticities were converted to mean residue molar ellipticities [θ] in units of deg × cm2/dmol. The sample of CP10A was prepared by adding a solution of perdeuterated DPC in 10 mm sodium phosphate buffer (pH 5) containing 10% D2O to the lyophilized peptide to give concentrations of 2 mm CP10A and 200 mmDPC-d38. The pH was adjusted to 4.0 (uncorrected for the isotope effect). NMR spectra were recorded at 37 and 42 °C on a Bruker AMX600 spectrometer operating at 600.13 MHz. Homonuclear TOCSY (21Braunschweiler L. Ernst R.R. J. Magn. Res. 1983; 53: 521-528Crossref Scopus (3108) Google Scholar), NOESY (22Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4838) Google Scholar), and DQF-COSY (23Rance M. Sørensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2597) Google Scholar) spectra were acquired. Water suppression was achieved using the WATERGATE technique (24Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3527) Google Scholar, 25Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Res. A. 1993; 102: 241-245Crossref Scopus (1112) Google Scholar) or by presaturation during the recycling delay (2–2.5 s). Spectra were collected with 512–800 data points in F1, 2000 data points F2, and 32–64 transients. TOCSY spectra were acquired using the Malcolm Levitt (MLEV)-17 pulse sequence (26Bax A. Davis D.G. J. Magn. Res. 1985; 65: 355-360Google Scholar) at a spin-lock time of 80 ms. NOESY spectra were recorded at mixing times of 70, 100, and 150 ms. Qualitative exchange rates of labile hydrogen atoms of CP10A in DPC micelles at 37 °C were determined by dissolving the lyophilized peptide in D2O containing DPC-d38 at a molar ratio of peptide to detergent of 1:100. The pH* was determined to be 3.9 (uncorrected for the isotope effect). Several one-dimensional NMR spectra were acquired during the first hour after dissolution in D2O. Then a TOCSY spectrum with a total acquisition time of ∼5 h was recorded followed by another one-dimensional spectrum to detect any slowly exchanging protons. The NMR data were processed with NMRPIPE (27Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar). Resolution enhancement was achieved by apodization of the free induction decay with shifted squared sine-bell window functions. The data were zero-filled to at least twice the size before Fourier transformation. Spectra were base-line-corrected using a 5th order polynomial function. All chemical shifts were referenced to internal 4,4-dimethyl-4-silapentane-1 sulfonate (DSS). All NMR spectra were analyzed using NMRVIEW version 4.0.5 (28Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2678) Google Scholar). NOE cross-peaks were integrated in the NOESY spectra acquired at 37 °C using a mixing time of 70 ms. The NOE volumes were converted to distances, which were calibrated using the average NOE volume of all resolved geminal methylene proton cross-peaks as well as cross-peaks between tryptophan aromatic ring protons. The HN-Hα distances thus calculated ranged from 2.65 to 2.99 Å, which is well within the covalently restricted range (2.3–3.1 Å). Each distance was converted to a distance restraint by calculating upper and lower distance bounds using the equations suggested by Hyberts et al. (29Hyberts S.G. Goldberg M.S. Havel T.F. Wagner G. Protein Sci. 1992; 1: 736-751Crossref PubMed Scopus (399) Google Scholar). Pseudo atom corrections were applied by adding 1 and 1.5 Å to the upper distance bound for unresolved methylene protons and methyl groups, respectively. Distance restraints involving resolved methylene protons were float-corrected by adding 1.7 Å to the upper bound. Structure calculations were performed using the distance geometry/simulated annealing programs DGII (Molecular Simulations Inc., San Diego, CA) and X-PLOR version 3.851 (30Kuszewski J. Nilges M. Brunger A.T. J. Biomol. NMR. 1992; 2: 33-56Crossref PubMed Scopus (210) Google Scholar, 31Nilges M. Clore G.M. Gronenborn A.M. FEBS Lett. 1988; 229: 317-324Crossref PubMed Scopus (772) Google Scholar, 32Nilges, M., Kuszewski, J., Brunger, A. T., Computational Aspects of the Study of Biological Macromolecules by NMR, Hoch, J. C., 1991, Plenum Press, New York.Google Scholar). Of 20 structures generated by DGII, 16 structures that had 3–6 NOE distance restraint violations of 0.1 Å were refined using X-PLOR and the CHARMM force field. The refinement consisted of simulated annealing, decreasing the temperature from 310 to 10 K over 50,000 steps (0.001 ps), resulting in 15 structures that converged with final energies of 25 ± 1 kcal/mol and 6 ± 1 NOE distance restraint violations ≥0.1 Å. The average largest NOE distance restraint violation was 0.20 ± 0.02 Å. Thin sections of CP10A-treated S. epidermidis and S. aureus were prepared to observe ultrastructural changes (Fig.1). The bacteria were treated with 10-fold MIC of CP10A for 10 min before fixing (20 μg/ml and 40 μg/ml for S. epidermidis and S. aureus, respectively). These electron micrographs showed the formation of intracellular lamellar membranes (mesosomes) in peptide-treated cells only. Often these mesosomes occurred around the septum. In contrast to the indolicidin-related peptide CP11CN-treated S. epidermidis cells (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar), no nuclear condensation was seen, and there appeared to be minimal cell wall effects with CP10A-treated cells. No apparent lysis or gross leakage of cellular cytoplasmic contents was observed. We have previously shown (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar) that CP10A had the same (Streptococcus hemolyticus and Corynebacterium xerosis) or 2–8-fold lower (S. aureus, methicillin-resistant Staphylococcus aureus, S. epidermidis, Enterococcus faecalis,Listeria monocytogenes, Streptococcus pyogenes) MICs compared with indolicidin against Gram-positive bacteria. For macromolecular synthesis studies, we determined the MICs of CP10A on the S. aureus wild-type strain (ATCC 25923) and the auxotrophic strain (ISP 67) in the presence of both LB and synthetic media. This was done to ensure that there are no significant differences between the two strains. The MIC of CP10A against the wild type was 2–4 μg/ml in LB and 2 μg/ml in the synthetic media. The MIC of CP10A against the auxotroph was 2 μg/ml in LB and 1 μg/ml in synthetic media. These results showed that there were no significant differences in activity against the two strains and in the two media. To determine if CP10A had intracellular effects, we performed standard macromolecular synthesis assays. S. aureus ISP67 was used in macromolecular synthesis experiments in the presence of 2- and 10-fold the MIC of CP10A. Killing assays done in conjunction with these experiments (Fig. 2) showed that at 2-fold MIC there were 50% or more survivors over the first 40 min, whereas at 10-fold MIC, there were less than 10% survivors in the same time frame. Nonetheless, there was no significant difference between the two concentrations in macromolecular synthesis effects (Fig.3), which demonstrated nearly complete inhibition. These results indicated that the effects on macromolecular synthesis at low peptide concentrations were probably not the result of dead or dying cells. Macromolecular synthesis of DNA, RNA, and proteins did not cease simultaneously, as expected if this was merely membrane disruption resulting in leakage of essential molecules. Histidine and uridine incorporation (Fig. 3, B and C) appeared to be affected first, with differences within the first 5 min after the peptide addition. Thymidine incorporation (Fig. 3 A) was not affected until after 10 min. These results revealed that CP10A had intracellular effects on S. aureus.Figure 3Effect of CP10A at 2-fold the MIC (triangles) and 10-fold the MIC (squares) on 3H-labeled thymidine (A), uridine (B), and histidine (C) incorporation into S. aureusISP67 DNA, RNA, and protein macromolecules. Diamonds, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine if CP10A had the ability to depolarize the cytoplasmic membrane ofS. aureus, we used the membrane potential-sensitive dye dipropylthiacarbocyanine (5Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (584) Google Scholar). CP10A almost completely depolarized the membrane at a concentration of 1 μg/ml (data not shown) within 5 min. These results were similar to those found with the α-helical peptides CP26 and CP29, which completely depolarized the membrane at low concentrations (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). In contrast, indolicidin and its derivative CP11CN never completely depolarized the cytoplasmic membrane of eitherS. aureus (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar) or E. coli (8Wu M. Maier E. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 7235-7242Crossref PubMed Scopus (631) Google Scholar). The conformation of CP10A was determined using NMR spectroscopy in the presence of DPC; therefore, it was important to establish that the structure of this peptide formed upon binding to detergent micelles was similar to the structure when bound to phospholipid bilayers, which more closely resemble the bacterial cytoplasmic membrane. Fluorescence and CD spectroscopy are suitable methods for comparing the interaction of peptides in different membrane environments. The fluorescence spectra of CP10A in aqueous solution and bound to different lipid and detergents are shown in Fig.4. The emission maximum of the peptide in buffer alone was around 355 nm. With all lipid environments, an 8-nm blue shift of the emission maximum as well as an increase in fluorescence intensity occurred, indicating a transfer of the tryptophan side chains from an aqueous to a hydrophobic environment (33Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar). The emission spectra in POPC, POPG, POPC:POPG (7:3), and DPC were all very similar, with peak wavelengths of around 347 nm, indicating that the environment experienced by the peptide in lipids and DPC was comparable. When the aqueous quencher KI was added in increments (data not shown), no change in fluorescence intensity was observed if the peptide was in the presence of liposomes, indicating that the tryptophan residues had become inaccessible to the aqueous buffer. Liposomes with incorporated spin-labeled lipids were used to estimate the average position of the tryptophan residues in the liposome, as spin labels will cause a decrease in tryptophan fluorescence when in close proximity. In the presence of head group labeled tempo-PC, there was an approximate 35% decrease in fluorescence. In the presence of 5-doxyl-PC and 12-doxyl-PC, there was ∼45 and 48% decrease, respectively. The absence of a significant difference between the effects caused by 5-doxyl-PC and 12-doxyl-PC may be explained by the presence of five tryptophan residues in CP10A. Some of the tryptophan side chains may have re
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