Dimerization of aurein 1.2: effects in structure, antimicrobial activity and aggregation of Ca ˆndida albicans cells

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ORIGINAL ARTICLE E. N. Lorenzo ´n • P. R. S. Sanches • L. G. Nogueira • T. M. Bauab • E. M. Cilli Received: 1 December 2012 / Accepted: 14 February 2013 / Published online: 22 March 2013  Springer-Verlag Wien 2013 Abstract Antimicrobial peptides (AMPs) are a promising solution to face the antibiotic-resistant problem because they display little or no resistance effects. Dimeric analogues of select AMPs have shown pharmacotechnical advantages, making these molecules promising candidates for the development of novel antibiotic agents. Here, we evaluate the effects of dimerization on the structure and biological activity of the AMP aurein 1.2 (AU). AU and the C- and N-terminal dimers, (AU)2K and E(AU)2, respectively, were synthesized by solid-phase peptide synthesis. Circular dichroism spectra indicated that E(AU)2 has a ‘‘coiled coil’’ structure in water while (AU)2K has an a-helix structure. In contrast, AU displayed typical spectra for disordered structures. In LPC micelles, all peptides acquired a high amount of a-helix structure. Hemolytic and vesicle permeabilization assays showed that AU has a concentration dependence activity, while this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action of AU. Notably, the antimicrobial activity against bacteria and yeast decreased with dimerization. However, dimeric peptides promoted the aggregation of C. albicans. The ability to aggregate yeast cells makes dimeric versions of AU attractive candidates to inhibit the adhesion of C. albicans to biological targets and medical devices, preventing disease caused by this fungus. Keywords Antimicrobial peptides  Aurein 1.2  Dimerization  Biological activity  Secondary structure Introduction Because of the drug resistance problem with traditional antibiotics, the development of novel antimicrobial agents becomes a very important challenge (Rossolini et al. 2007). A potential solution may lie in antimicrobial peptides (AMPs), because they have a broad-spectrum activity, act rapidly and rarely develop drug resistance (Dennison et al. 2007; McCubbin et al. 2011; Kamysz et al. 2006; Pini et al. 2012). AMPs are found in animals, plants, insects, and microorganisms, forming part of the innate defense systems (Peters et al. 2010; Ribeiro et al. 2011; Cespedes et al. 2012; Libe ´rio et al. 2011; He et al. 2013). Most of the AMPs reported disrupt the membrane of cells via three general mechanisms. In the first, the peptides remain tightly bound to the membrane interface until reach a threshold concentration when promote bilayer damage via detergent or carpet-like mechanism. In this mechanism, peptides do not necessarily insert into the hydrophobic membrane core. On the other hand, peptides could form transmembrane pores. There are two pore-forming mechanisms. In the ‘‘barrel-stave’’ model, the peptides adopt a helical conformation and aggregate into a barrel-like structure that spans the membrane with the peptides lying perpendicular to the plane of the membrane. In an alternative model, named ‘‘toroidal pore’’, the peptides are tightly bound to the polar lipid groups of the membrane, promoting the bending of the bilayer (Ambroggio et al. Electronic supplementary material The online version of this article (doi:10.1007/s00726-013-1475-3) contains supplementary material, which is available to authorized users. E. N. Lorenzo ´n  P. R. S. Sanches  E. M. Cilli (&) Institute of Chemistry, University Estadual Paulista Araraquara (UNESP), Araraquara, SP, Brazil e-mail: cilli@iq.unesp.br L. G. Nogueira  T. M. Bauab Faculty of Pharmaceutical Sciences, University Estadual Paulista (UNESP), Araraquara, SP, Brazil 123 Amino Acids (2013) 44:1521–1528 DOI 10.1007/s00726-013-1475-32005; Fernandez et al. 2009; Castro et al. 2006). In addition to the membrane disrupting mechanisms described above, several studies indicate that cell membrane permeabilization alone may not be enough to explain peptide antimicrobial activity. The observations of peptide-induced lipid clustering of anionic components from zwitterionic lipids led to the introduction of the ‘‘lipid clustering mechanism’’ (Epand et al. 2010; Teixeira et al. 2012). Parameters, such as charge, amphipathicity, hydrophobicity and helicity have been extensively studied and correlates directly with the biological activity of AMPs (Matsuzaki 2009; Zhu et al. 2007; Huang et al. 2010). In addition, the knowledge that the aggregation of molecules of AMPs, either before or after binding to the membrane surface, is a prerequisite to pore formation, led researchers to the design of dimeric peptides. (Sengupta et al. 2008; Matsuzaki 2009; Pini et al. 2005; Hornef et al. 2004; Lee et al. 2008; Welling et al. 2007; Zhu and Shin 2009a). In this way, several bioactive sequences were dimerized obtaining pharmacotechnics advantages, like enhanced antimicrobial activity, solubility and proteases resistant (Hara et al. 2001; Dempsey et al. 2003; Pini et al. 2005; Dewan et al. 2009; Liu et al. 2010). However, the effect of this modification is unclear since dimeric versions of some AMPs lost antimicrobial activity or gained toxicity (Yang et al. 2009; Zhu and Shin 2009b; Lorenzo ´n et al. 2012). Here, we used the AMP Aurein 1.2 (AU) as the framework to study the effects of dimerization on its structure and biological activity. In this way, AU and the C- and N-terminal dimeric version (AU)2K and E(AU)2, respectively, were synthesized. This is the first study on the effects of dimerization on a peptide proposed acting by carpet-like mechanism. Materials and methods Chemicals and microorganisms Only analytical grade reagents from commercial suppliers were used. All solutions were prepared with Milli-Q water (Millipore Reagent Water System, USA). Solvents for chromatographic procedures were of HPLC grade from several sources. 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids and resins were purchased from Synbiosci (USA) and Novabiochem (USA). Solvents and reagents for peptide synthesis were from Sigma Aldrich Co. (USA) and Fluka (Switzerland). DCM (dichloromethane) and DMF (dimethylformamide) were purchased from Hexis Cientifica (Brazil). All lipids and surfactants were purchased from Avanti Polar Lipids (USA). We used the bacterial strains Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) and the yeast Candida albicans (ATCC 18804). Peptide synthesis All peptide syntheses were manually performed by solidphase peptide synthesis (SPPS) using the standard Fmoc (9-fluorenylmethyloxycarbonyl) protocols on a RinkMBHA resin. For the C-terminal dimer, Fmoc-Lys(Fmoc)- OH was attached to the resin, and after a- and e-Fmoc group deprotection with 20 % piperidine/dimethylformamide (DMF), the two chains of AU were simultaneously elongated (Fig. 1). On the other hand, after the synthesis of AU, Boc-Glu-OH, without side chain protector, was used to link two chains of this peptide, obtaining the N-terminal dimer (Fig. 1). In all cases, the amino acids were coupled at twofold excess over the amino component in the resin, using diisopropylcarbodiimide (DIC)/N-hydroxybenzotriazole (HOBt) in 50 % (v/v) DCM (methylene chloride)/ DMF. After 2 h, the qualitative ninhydrin test was performed to estimate the completeness of the coupling reaction; the recoupling procedure was done when the ninhydrin test was positive. Cleavage of the Rink MBHA resin and removal of the side chain protecting groups for all the peptides were simultaneously performed with 95 % TFA, 2.5 % water and 2.5 % TIS for 2 h. After this procedure, the crude peptides were precipitated with anhydrous ethyl ether, separated from soluble nonpeptide material by centrifugation, extracted into 0.045 % (v/v) TFA/H2O (solvent A), and lyophilized. The crude peptides were dissolved in solvent A and purified by semi-preparative HPLC on a Beckman System Gold using a reverse-phase C18 column with a linear gradient 40–80 % of solvent B (0.036 % (v/v) TFA/acetonitrile) for 120 min. The flow rate was 5 mL/min. UV detection was carried out at 220 nm. The peptide homogeneity was checked by analytical HPLC on a Shimadzu system, using solvents A and B with a linear gradient of 5–95 % (v/v) of solvent B for 30 min, at a flow rate of 1.0 mL/min and UV detection at 220 nm. The identity of the peptide was confirmed by mass spectrometry in positive ion mode ESI on a Bruker model apparatus (Germany). Antimicrobial assay The minimum inhibitory concentration (MIC) was performed following the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2006; CLSI 2008). The antibacterial and antifungal activity tests were performed using the broth microdilution method. In brief, bacterial cells in Mueller–Hinton media (aliquots of 80 lL containing 1.5 9 107 CFUs) were incubated with a serial dilution (128–1 lmol/L) of the synthetic peptides dissolved in Milli-Q water. After incubation for 24 h at 37 C, the microtiter plates were analyzed visually by the addition of rezasurine. For the antifungal assay, the medium used was 1522 E. N. Lorenzo ´n et al. 123RPMI 1640 with L-glutamine buffered to pH 7.2 with 0.165 M morpholinepropanesulfonic acid (MOPS), supplemented with 2 % glucose. The cell suspension (final concentration of 1 9 103 to 2.5 9 103 CFUs/mL) was inoculated on a microdilution plate previously prepared with the synthetic peptides diluted in a concentration range from 128 to 1 lmol/L. The plates were incubated at 37 C for 48 h. Each assay was performed in triplicates. For control, bacterial suspensions were incubated either in Milli-Q water. In addition, control drugs used were amphotericin B. and fluconazole. The MIC was defined as the lowest concentration of the peptide or other antimicrobial agent at which no growth was detectable. Vesicle permeabilization Large unilamellar vesicles (LUVs) composed of 95 % DPPC (1,2-dipalmitoyl-3-phosphocholine) and 5 % DPPA (1,2-dipalmitoyl-3-phosphatidic acid) were prepared by mixing the appropriate amounts of lipid in a 4:1 chloroform:methanol mixture in a round-bottom flask. The solvent was rapidly evaporated using nitrogen gas. The lipid biofilm was placed under a vacuum overnight and hydrated at 60 C with 80 mmol/L of CF in Tris (0.01 mol/L, pH 7.4) and NaCl (0.15 mol/L) to give a final lipid concentration of 15 mmol L-1. This suspension was extruded 40 times through two stacked nucleopore polycarbonate filters (100 nm pore size) using an extruder system from Avanti Polar Lipids (USA) at approximately 40 C. Vesicles were separated from non-encapsulated CF by gel filtration on a Sephadex G-50 column using Tris (0.01 mol/L, pH 7.4) and NaCl (0.15 mol/L) for elution. The release of carboxyfluorescein (CF) from vesicles was measured by the fluorescence intensity at a wavelength of 520 nm (492 nm excitation wavelength) after the addition of 1, 4 and 16 lmol/L of peptide. Data were acquired using a fluorescence spectrophotometer (Cary Eclipse, VARIAN). The experiments were performed at 25 C in triplicate. Hemolysis assay Hemolysis assays were performed using the experimental procedure described by Castro et al. (Castro et al. 2009). In brief, before use, freshly human red blood cells (RBCs) was washed three times with 0.01 mol/L Tris–HCl pH 7.4 containing 0.15 mol/L NaCl (Tris–saline). A suspension of 1 % (v/v) erythrocytes was made with packed red blood cells re-suspended in Tris–saline. Synthetic peptides were dissolved in Tris–saline at an initial concentration of 128 mmol/L, and were serially diluted in the same buffer to determine its HC50 (concentration that causes 50 % hemolysis). As a positive control (100 % lysis), a 1 % (v/v) Triton X-100 solution was used. After incubation for 1 h at 37 C, the samples were centrifuged at 1,000g for 5 min. Aliquots of 100 lL of the supernatant were transferred to 96-well microplates, and the absorbance was determined at 405 nm. The assay was performed in triplicate. CD spectra Circular dichroism spectra were obtained between 190 and 250 nm with a JASCO J-715 CD spectrophotometer (Japan) on nitrogen flush in 1 mm path length quartz cuvettes at room temperature. The peptide concentration was 60 lmol/L. To investigate the conformational changes by membrane environments, a solution containing 10 mmol/L of lysophosphatidylcholine (LPC) was used. CD spectra were typically recorded as an average of six scans that were obtained in millidegrees and converted to molar ellipticity [h] (in deg cm2 dmol-1). Statistics MIC values are reported as the mode of a set of three independent assays. Hemolysis data are reported as the mean of a set of three independent assays. Fig. 1 Strategy of peptides synthesis Dimerization of aurein 1.2 1523 123Results Peptide design and synthesis The monomeric and two dimerics versions of the AMP AU 1.2 were synthesized using the SPPS (Fig. 1). The peptides were obtained with a high level of purity (above 98 %) and the identities of these molecules were confirmed by electrospray mass spectrometry (Table 1). Biological activity The peptides were evaluated in term of antimicrobial, antifungal and hemolytic activity. Dimerization of AU decreases the ability to inhibit the growth of bacteria and fungi (Table 2). The MICs values of N- and C-dimers were bigger than monomer. However, dimeric peptides promoted the aggregation of C. albicans cells (Fig. 2). To evaluate the toxicity of the peptides, we used the hemolysis test. The results summarized in Fig. 3 show that E(AU)2 has no hemolytic activity, while (AU)2K caused 10–30 % hemolysis in the range of concentration used. On the other hand, the monomer showed the bigger HC50 values. Vesicle permeabilization CF release from LUVs composed of DPPA/DPPC (5/95 %) was evaluated in three different peptide concentrations. The results showed that the addition of peptides promote the release of the dye, as indicated by an increase in fluorescence (Fig. 4). In addition, AU has a concentration dependence permeabilization activity, whereas this effect was less pronounced for dimeric versions. CD spectroscopy CD spectroscopy studies in water showed that (AU)2K has a typical a-helix spectrum with double maxima at 208 and 222 nm. The ellipticity radio (222/208 nm[ 1) for E(AU)2 determined a ‘‘coiled coil’’ structure where two helices wrap around each other (Bromley and Channon 2011). In contrast, AU displayed typical spectra for disordered structures. For all the peptides, an a-helical structure was induced by the non-polar environment of LPC micelles (Fig. 5). Discussion Several studies have shown that dimerization of AMPs improves antimicrobial activity, solubility and proteases resistant (Falciani et al. 2007; Lee et al. 2008; Taylor et al. 2007; Welling et al. 2007; Dempsey et al. 2003; Hara et al. 2001). Here, we study the effects of dimerization in structure and biological activity of the AMP AU 1.2. This 13-residue peptide is the most studied member of the aurein0s family (Giacometti et al. 2007; Karbalaeemohammad and Naderi-Manesh 2011; Dennison et al. 2007; Lee et al. Table 1 Results of peptide synthesis Peptide Yield (%) Retention time (min) Mass (Da) Observeda Calculated AU 27 20.1 1,479.9 1,479.8 (AU)2K 6 24.9 3,070.7 3,070.2 E(AU)2 2 24.0 3,071.5 3,070.7 a Obtained from mass spectra of the purified peptides Table 2 Antimicrobial activities of the synthetic peptides Peptı ´deo MIC (lmol/L) E. coli S. aureus C. albicans AU 16 8 32 (AU)2K 128 [128 [128 E(AU)2 128 [128 [128 Concentration(µmol/L) AU (AU)2K E(AU)2 128 64 32 16 8 4 Fig. 2 Aggregation of C. albicans cell induced by dimeric analogues 0 10 20 30 40 50 60 128 64 32 16 8 4 % Hemolysis Concentration (µmol/L) E(AU)2 (AU)2K AU Fig. 3 Hemolytic activities of the synthetics peptides 1524 E. N. Lorenzo ´n et al. 1232010; Li et al. 2006). Previous studies have proposed that this peptide acts by a carpet-like mechanism (McCubbin et al. 2011; Cheng et al. 2009; Ambroggio et al. 2005; Chen and Mark 2011). It is supported by the fact that it is too short to span the membrane. We decided to use this AMP as the scaffold because there is no information about the effects of dimerization of AMPs described as having the carpet-like mechanism. In addition, many studies have shown that the modifications of C- and N-terminus of AMPs are important to biological activity (Crusca et al. 2011; Sforc ¸a et al. 2004). Sforc ¸a et al. (2004) showed that the amidation of C-terminus promotes a structural perturbation of the amphipathic a-helix and affects its biological activity. In addition, Crusca et al. (2011) demonstrated that the charge of N-terminal is important to the structure and selectivity of the antimicrobial peptide Hyl-A1. Taking it into consideration, we designed the C- and N-terminal dimeric versions of AU to evaluate the structure and the biological activity of these analogues. As shown in Table 2, dimerization decreases the ability of the peptide to inhibit the growth of bacteria and fungi. These results are in agreement with Yang et al. (2009) and Ma ¨ntyla ¨ et al. (2005) whose have shown negative effect with dimeric versions of PST13-RK and Temporin A, respectively. However, others studies with dimeric forms of AMPs have shown pharmacotechnical advantages, such as enhanced antimicrobial potency, solubility, and resistance to proteases (Hara et al. 2001; Dempsey et al. 2003; Pini et al. 2005; Dewan et al. 2009; Liu et al. 2010). These controversial results show that the effects of dimerization of AMPs needs to be better studied. In antifungal studies, the dimeric version of AU promoted the aggregation of C. albicans cells (Fig. 2). This may be due to the interaction of the peptides with yeast cell wall carbohydrates. Tsai et al. (2011) showed that the peptide LL-37 interact with mannans, the main component of the C. albicans cell wall, promoting the aggregation of these cells. It has been reported also that AMPs interact with lipopolysaccharides and lipoteichoic acid, the distinct components of the gram-negative and gram-positive bacteria, respectively (Bucki and Janmey 2006; Giacometti et al. 2006). Previous studies have shown that dimeric peptides may be inhibited from passing through the cell walls of prokaryotic cells, (Jiang et al. 2011; Lorenzo ´n et al. 2012). These interactions with cell wall components could explain the lower capacity of dimeric AMPs to reach the membrane of bacteria and fungi. Hemolytic activity is always a potential barrier preventing the use of AMPs as systemic therapeutics. To evaluate the toxicity of the peptides, we used the hemolysis test, the most commonly procedure to characterize the eukaryotic membrane damage caused by AMPs. The results summarized in Fig. 3 shows that dimeric peptides have little to no hemolytic activity and seem to be not concentration dependent. In contrast, AU has a concentration dependence activity above 32 lmol/L. CF release from LUVs also showed that AU has a concentration dependence permeabilization activity, whereas this effect was less pronounced for dimeric versions (Fig. 4). 0 2 4 6 8 10 0 20 40 60 80 100 120 140 Intensity (a. u.) Time (min) AU 1 umol/L (AU)2K 1 umol/L E(AU)2 1 umol/L 0 2 4 6 8 10 0 20 40 60 80 100 120 140 Intensity (a. u.) Time (min) AU 4 umol/L (AU)2K 4 umol/L E(AU)24 umol/L 0 2 4 6 8 10 0 20 40 60 80 100 120 140 Intensity (a. u.) Time (min) AU 16 umol/L (AU)2 K 16 umol/L E(AU) 216 umol/L a b c Fig. 4 Carboxyflourescein release. Peptide concentration: 1 (a), 4 (b) and 16 lmol/L (c) Dimerization of aurein 1.2 1525 123These results are explained by the aggregation among peptide chain of the dimeric version. This hypothesis was confirmed by CD studies (Fig. 5). Vesicles used in CF release have no cell wall components, including carbohydrates, to interact with the peptides. This data showed that the difference in biological activity is not only due the peptide-cell wall interactions. To evaluate the correlation among the above results and the structure of the peptides, CD spectroscopy was used. When compared with AU, dimeric versions have more defined secondary structures in aqueous solution. C-terminal dimerization has induced a-helix structure while N-terminal dimerization promotes an interaction between the two peptide chains, leading to a ‘‘coil-coiled’’ structure. It is well known that the first step in any mechanism of action is the electrostatic attraction of the cationic peptide to the anionic membrane. In general, peptides are in a random coil conformation in solution but adopt an a-helical conformation when associated with the lipid membrane, the second step in the peptide-membrane binding process (Seelig 2004). Our results indicated that the structured-state in solution imposed by dimerization is more definite and it may modify the capacity of the peptide to bind the membrane. Besides that, in the presence of LPC micelles, all peptides acquired the same content of a-helical conformation, as indicated by one positive band at 190 nm and negative bands in 208 and 222 nm. These results indicated that the difference in biological activity among monomeric and dimeric version of AU occurs mainly before (with cell wall component) or in the first step of the interaction with the phospholipid bilayer. Conclusion The analysis of the whole data indicates that dimerization of AU decrease the antimicrobial activity. In this study, two factors could explain these data. The first is the interaction of AMP with cell wall components, as confirmed by the aggregation of C. albicans cells. The other is due to the structure-state in solution that promotes different initial interaction with the membrane and could change the mechanism of action. In addition, the ability of aggregating yeast cells, make the dimeric versions of AU future drug candidates to prevent C. albicans adhesion to biological targets and medical devices, such as prostheses and catheters, preventing disease caused by this fungus. Acknowledgments The authors are grateful to Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico (CNPq), Fundac ¸a ˜o ao Amparo a Pesquisa do Estado de Sa ˜o Paulo (FAPESP), Coordenac ¸a ˜o de Aperfeic ¸oamento de Nı ´vel Superior (CAPES) for financial support. EMC is senior researchers of the CNPq, and ENL was the recipient of Ph.D fellowship from CNPq. 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