Membrane Selectivity of Antimicrobial Peptides - ACS Symposium

Chapter 4

Membrane Selectivity of Antimicrobial Peptides CharleneM.Mello and JasonW.Soares

Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: May 30, 2008 | doi: 10.1021/bk-2008-0984.ch004

Macromolecular Science Team, Natick Soldier Research Development and Engineering Center, Natick Massachusetts

Antimicrobial peptides are ubiquitous, classified primarily based upon secondary structure, and often possess broad spectrum activity. Our laboratory investigates the class of amphipathic α-helical peptides with an aim to elucidate the principles that determine peptide selectively for bacterial membranes. Here we will review the current state of knowledge surrounding peptide characteristics that influence selective bacterial activity and present new data demonstrating Gram selective binding. Furthermore, we suggest that tailoring the binding affinity and selectivity of native peptides to capture and detect pathogenic cells will be realized in the near future.

52

U.S. government work. Published 2008 American Chemical Society.

In Microbial Surfaces; Camesano, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

53


Background Antimicrobial peptides are part of an innate immune defense system in all organisms, fighting against microbial infection and ensuring survival in an everchanging environment (1). Hundreds of antimicrobial peptides have been identified which are generally classified based upon their secondary structure (2). P-sheet peptides with one or more disulfide bonds (3), and a-helical peptides (4) comprise the majority of the peptides investigated in the current literature. Despite their differing conformations, most form an amphipathic structure with a distinct hydrophobic and cationic face in membrane environments. In general, they exhibit broad spectrum activity against Gram-positive and Gram-negative bacteria, fungi, and viruses. However, peptides with similar structures can possess rather different activity profiles. Initial binding to target cells does not involve specific receptors on the cell membrane, but non-specific electrostatic interactions between the positively charged residues of the peptide and the anionic membrane (5). Due to the non specific nature of the interaction, many antimicrobial peptides also lyse red blood cells and are cytotoxic to mammalian cells. Model phospholipid membranes have been studied to understand the mechanism of cytotoxicity. Peptides that bind preferentially to bacterial membranes efficiently disrupt the packing of anionic phospholipids while zwitterionic phospholipids are only permeated by peptides active upon mammalian cells (6). Lipid chemistry and peptide properties are collectively responsible for preferential bacterial activity. This chapter will summarize investigations focused upon peptide characteristics that may define bacterial selectivity.

Principles for Bacterial Membrane Specificity A n area of intense investigation has been focused on structure/function relationships of native, truncated, and rationally designed peptides to enhance antimicrobial activity while reducing hemolysis. Based upon these numerous reports, a few overarching principles are emerging. A well controlled balance of peptide structure, charge, hydrophobicity, flexibility, and assembly state appear to be important for selective binding and activity towards bacterial cell membranes relative to eukaryotic membranes.

Peptide Length and Assembly State To explore the impact of self-assembly on bacterial selectivity, Glukhov et al. studied a family of cationic antimicrobial peptides in SDS micelles and



54 phospholipid vesicles capable of inserting into anionic lipid membranes but not zwitterionic membranes. The observed binding to model membranes, high antimicrobial activity and low hemolytic activity were attributed to peptide dimerization. SDS resistant dimers were confirmed by gel electrophoresis and the presence of such peptide dimers in anionic membranes revealed by fluorescence resonance energy transfer. The authors suggest that the sequence motif, A X X X A where X is any amino acid, is responsible for promoting dimerization, and possibly higher oligomerization states (7). It is not clear from this study, however, whether dimer formation occurs in aqueous conditions or following membrane binding Peptide length and preassembly state of cationic antimicrobial peptides were investigated by Sal-Man and co-workers to determine i f these are important discriminating factors for bacteria and erythrocytes. Lysine/Leucine rich peptides with variable lengths (13, 16, and 19 amino acids long) and covalently linked pentameric peptide bundles were evaluated for their activity and binding affinity to model membranes. The antibacterial activity of the monomeric peptides increased as a function of peptide length while activity towards erythrocytes remained constant. However, the activity of all bundles was not dependent upon peptide length. Preassembly increased activity towards erythrocytes which is supported by an observed 300-fold increase in peptidemembrane binding affinity toward zwitterionic vesicles compared with negatively charged vesicles. Therefore, preassembly of pentameric structures results in a loss of bacterial selectivity (8). Similarly, a single amino acid substitution in the leucine zipper motif of melittin, which has a propensity for oligomerization, results in a dramatic reduction of hemolytic activity but not antimicrobial activity. Disruption of oligomerization in high ionic strength conditions as well as membrane environments was demonstrated (9). Cooperatively, these results suggest that bacterial selectivity may require peptides to be monomeric and linear prior to membrane binding, but have the propensity to self-assemble in bacterial membrane environments. Furthermore, the composition of bacterial membranes may be more favorable to oligomerization of selective peptides than the zwitterionic lipids.

Hydrophobicity and Charge Sequence analogs of pseudin-2, a naturally occurring 24 amino-acid amphipathic ct-helical antimicrobial peptide, were utilized to explore the role of cationicity and hydrophobicity in bacterial selectivity. Neutral and acidic amino acid residues were increasingly substituted on the hydrophilic face of the a-helix with the cationic amino acid, L-lysine. A n increase in activity towards a number of Gram-negative and Gram-positive bacteria while retaining low hemolytic activity was observed. Increasing the number of lysine residues in the



55 hydrophilic face to 4 and 5; however, did not further enhance antimicrobial potency but did increase hemolytic activity (10). Alternatively, modifying the hydrophobic face of antimicrobial leucine zippers by substituting leucine residues with alanine lowered the hemolytic activity while maintaining similar secondary structure; suggesting that minimizing hydrophobicity while maintaining amphilicity may preserve bacterial selectivity (9,11). Finally, molecular dynamic simulations were used to explore the effect of charge at the C-terminus of protegrin-like peptides on activity and toxicity (12). Removing a positively charged residue at the C-terminus does not significantly alter the peptide's toxicity, but replacing the positive charge with a negative charge reduces mammalian toxicity. These results hold great promise to rapidly advance the field by coupling simulations with model membrane experimental studies predicting both activity and selectivity.

Central Hinge or Helix Flexibility IsCT is a natural amphipathic a-helical peptide with high activity against both mammalian and bacterial cells. Lee et al designed several novel peptide analogs with bacterial cell selectivity based on the IsCT peptide sequence. Solution structures were determined by 2 D - N M R spectroscopy and revealed that three peptides exhibiting the greatest selectivity also had a central hinge structure (13). A similar conclusion by this group was obtained by investigating a lysine/leucine peptide possessing limited selectivity (14). Sequence variants of these peptides were designed with proline substitutions to evaluate the impact of proline-induced bends on activity. Substitution of a central leucine residue yielded strong antibacterial activity but had no detectable hemolytic activity. Furthermore, proline substitutions in the hydrophobic face resulted in a much greater reduction of hemolytic activity than substitution in the hydrophilic face and D-proline substitutions improve bacterial selectivity more than L-proline (15). It is unlikely, however, that these substitutions have generated a bend in the structure; circular dichroism revealed a dramatic reduction in helix content (50% to 10%) suggesting a significant disruption of the helix. Nonetheless these results suggested that a central proline and a-helical content contribute to bacterial cell selectivity. Shin et al explored the role of two central glycine residues in pleurocidin. The tertiary structure determined by N M R spectroscopy revealed that pleurocidin has a flexible structure between the long helix from Gly3 to G l y l 7 and the short helix from G l y l 7 to Leu25 (16). Alanine substitutions at these central glycine residues (pleurocidin-AA) yielded antibacterial activities similar to pleurocidin but had dramatically increased hemolytic activity (17). The non-cell-selective antimicrobial peptide, pleurocidin-AA, interacted strongly with both negatively charged and zwitterionic phospholipid membranes.



56 Furthermore, the a-helical content of the peptide variant when bound to SDS micelles increased dramatically and is consistent with the removal of a hinge region. Shin et al also demonstrated that an insertion of a Gly-Pro sequence into dermaseptin S3 resulted in a drastic reduction in hemolytic activity, with retention of the antibacterial activity (18)\ presumably due to a proline induced bend. Bend or hinge regions of peptides have also been resolved with molecular dynamics simulations. A n 8-residue helical antimicrobial peptide ovispirin-1 and its analogs novispirin-GlO and novispirin-T7 in SDS micelles were predicted to contain bend or hinge regions and are in good agreement with membrane-bound peptide structures predicted by solid state N M R (19). Collectively, the studies presented here suggest that structural flexibility between two helixes may play a key role in bacterial cell selectivity of antimicrobial peptides.

Methodology Elucidation of Binding Selectivity Contrary to the literature summary presented here, which focuses upon activity based selectivity, our research centers around the determination of native binding affinity and varied binding selectivity for Gram-negative and Grampositive bacteria to improve our understanding of the mechanisms of peptide discriminatory behavior. To probe peptide binding behavior, a whole cell binding assay (Figure 1) was developed utilizing antimicrobial peptides synthesized via F M O C solid-phase peptide synthesis with the addition of a cterminal cysteine for site-directed immobilization onto a maleimide reactive microplate. Whole bacterial cells (e.g. Escherichia coli 0157:H7, S. aureus) grown to mid-log phase (OD oo=l) were harvested, washed and resuspended in phosphate buffered saline, pH 7.2 (10 cfu/ml). The cell suspension is added to the wells (10 cfu/well) containing immobilized peptide. Peptide binding for the respective cells is evaluated by adding a horseradish peroxidase (HRP)conjugated polyclonal antibody for the target cell. A 2-component T M B peroxidase substrate system is then added and absorbance at 650nm after 30minute incubation is recorded. 6

8

7

Antibody Normalization To accurately depict peptide binding for the whole cells, the binding data must be normalized; compensating for antibody affinity differences for their



Bind whole bacterial cells to immobilized peptide

Bind a peroxidase labeled antibody specific to the bacterial cells

Color development to determine the cell binding ability of the peptide

Figure 1. Schematic of whole cell binding assay. Whole cells are grown to mid-log and bound to the immobilized peptides. Peptide binding for the whole cells is evaluated with horseradish peroxidase (HRP)-conjugatedpolyclonal antibodies. Color development was measured at absorbance of650nm after 30 minutes incubation.

Prepare microplate wells by immobilizing cys-derivatized peptide



58 respective target cells. At an identical number of cells, each antibody (Horseradish-peroxidase conjugated anti-£. coli 0157:H7, anti-S. aureus (general species), and anti-£. coli (general species)) produced significantly different responses for their respective cells (Figure 2). It is not currently possible to quantitate the number of cells bound per peptide; therefore, correction formulas were determined to correlate initial peptide binding affinity for E. coli ML35 and S. aureus to a corrected value. The corrected value is relative to an effective normalization of E. coli ML35 ( A T C C 43827) and S. aureus 27217 ( A T C C 27217) antibody affinity to that of E . coli 0157:H7 ( A T C C 43888). The antibody affinity curves were fit exponentially and linearized. Curves were fit with linear equations and analyzed at X 0 1 5 7 X M U S X S. aureus where X = log (cells bound). Subsequent linearization results in the following linear equations: =

=

In yoi = 1.057Xoi57-7.326 57

In y L35= 1 .742Xml35 -14.670 M

(1)

(2)

Solving for number of cells (X),

yoi57 + 7.326) / 1.057

(3)

= (In y 35 + 14.67) / 1.742

(4)

X0157 = O n

X

M L 3 5

ML

Evaluate at equal X ,

(In yoi57 + 7.326) / 1.057 = (In

y M L 3

5 + 14.670) / 1.742

In yoi5? = 0.607 In y L3s(org) + 1.579 M

(5)

(6)

Translation of the corrected antibody curves enables the direct correlation of the E. coli ML35 ( V M U S ) and S. aureus (ys.aureus) with E. coli 0157:H7 absorbance ( V 0 1 5 7 ) and thus the development of E. coli ML35 and S. aureus 27217 peptide binding affinity correction formulas (Table I). These correction formulas allow for accurate depiction of a peptide's relative binding affinity for S. aureus and E. coli ML35. This antibody normalization method can be implemented for any combination of bacterial cells and relevant antibodies.


59


— E c o l i 0157:H7 • S. aureus 27217 — 6 — E coli ML35

\ - 2.50

BP——•—^-

*

Log cells (cfu/ml)

Figure 2. Antibody affinity curves ofAnti-E. coli 0157.H7, Anti-S. aureus (gen spec), andAnti-E. coli (gen spec) for E. coli 0157.H7, S. aureus 27217, andE. coli ML35 whole cells respectively.

Table I. Antibody Correction Formulas Bacteria E. coli ML35 S. aureus 27217

Correction formula In

y L35(cotT) M

= 0.607 In

y L35(or ) M

g

+ 1.579

=

In ys.aureus(corr) 0.657 In ys.aureus(org) + 0.156 y = initial peptide binding affinity for the respective cell. y = corrected peptide binding to compensate for relative differences in respective antibody affinities. Both are in measures of absorbance (Abs 650nm) org

corr


60


Antimicrobial Peptide Binding To select peptide candidates for investigation, an antimicrobial peptide database was developed classifying peptides based on structural characteristics, antimicrobial activity, and amino acid sequence. Downselection was based on a lack of post-translational modifications, absence of cysteine residues, and activity for Gram-negative E. coli. The selection criteria were motivated by the need for rapid chemical synthesis and the desire to control peptide orientation during immobilization for subsequent whole cell binding assays. Cecropin PI isolated from a pig intestinal parasitic nematode (Ascaris suum) (21), vector mosquito cecropin A (Aedes albopictus) (23), winter flounder pleurocidin (Pleuromectes americanus) (20), African clawed frog P G Q (Xenopus laevis) (22), medfly ceratotoxin A (Ceratitis capitata) (24), and sheep SMAP-29 (Ovis aries) (25) were all chosen for this study. The full-length peptides were evaluated for binding of E. coli 0157:H7 relative to non-pathogenic E. coli ML35 and Gram-positive S. aureus (Table II). A l l six of the peptides exhibited preferential binding for the Gram-negative E. coli 0157.H7 relative to the Grampositive 5. aureus. Cecropin PI and P G Q also exhibited preferential binding for the pathogenic E. coli 0157.H7 relative to non-pathogenic E. coli.

Conclusions Despite a significant amount of experimental data centered around molecular mechanisms and characteristics of peptide-mediated cell lysis, degree of peptide insertion into the membranes, the extent and significance of pore formation, membrane destabilization process and other studies; few reports have focused on the the selectivity of these peptides. The limited studies in current literature suggest that bacterial selective antimicrobial activity may be driven by several factors including assembly state, hydrophobicity, charge, and flexibility. Peptides which favor anionic model membranes may require a monomeric state prior to membrane binding. Increasing charge in the hydrophilic face can improve selectivity as well as minimizing hydrophobicity while maintaining the amphipathic structure. In addition, structural flexibility particularly in the center of the molecule appears to play a role in minimizing mammalian toxicity while maintaining antimicrobial activity. Finally, we have demonstrated that antimicrobial selectivity can be extended beyond the eukaryotic/prokaryotic discrimination to Gram specific binding of selected peptides. These results suggest that tailoring peptide sequence may yield improved selectivity for pathogen specific capture and destruction.


61 Table II. Antimicrobial peptide discriminatory capability Antimicrobial peptide


pleurocidin cecropin p i PGQ ceratotoxin A cecropin A SMAP-29

E. coli 0157.H7 binding? 1.256 2.026 1.934 1.252 1.069 1.334

E. coli ML35 binding 0.984 1.205 1.055 0.925 0.822 1.087

b

0

n

UGram neg.

1.276 1.681 1.833 1.354 1.300 1.227

S. aureus n 27217 ^Qrampos. binding 16.526 0.076 0.217 9.336 8.954 0.216 5.069 0.247 0.140 7.636 0.179 7.453 d

"Absorbance values at 650nm ^corrected absorbance values at 650nm for E. coli ML35 (In y M L 3 5 c o r r 0-607 In + 1.579) and for S aureus 27217 (In y w ^ c o r r ) = 0.657 In ywe^org) + 0.156) DGram = antimicrobial peptide discriminatory binding capability for E. coli 0157:H7 relative to a non-pathogenic Gram negative E. coli ML35 =

VML35

c

neg

=

antimicrobial peptide discriminatory binding capability for E. coli 0157:H7 relative to Gram-positive S. aureus

^Gram-pos.

References 1. 2. 3.

Boman, H. G. Annu. Rev. Immunol. 1995, 13, 61-92. Nicolas, P.; Mor, A . Annu. Rev. Microbiol. 1995, 49, 277-304. Lehrer, R. I.; Lichtensien, A . ; Ganz, T. Annu. Rev. Immunol. 1993, 11, 105128. 4. Tossi, A . ; Sandri, L.; Giangaspero, A . Biopolymers 2000, 55, 4-30. 5. Shai, Y. Biochim. Biophys. Acta 1999, 1462, 55-70. 6. Papo, N.; Shai, Y. Peptides 2003, 24, 1693-703. 7. Glukhov, E.; Stark, M.; Burrows, L . L.; Deber, C. M. J. Biol. Chem. 2005, 280, 33960-7. 8. Sal-Man, N.; Gren, Z.; Shai, Y. Biochemistry 2002, 41, 11921-30. 9. Asthana, N.; Yadav, S. P.; Ghosh, J. K . J. Biol. Chem. 2004, 279, 55042-50. 10. Pal, T.; Sonnevend, A . ; Galadari, S.; Conlon, J. M. Regul. Pept. 2005, 129, 85-91. 11. Ahmad, A . ; Yadav, S. P.; Asthana, N.; Mitra, K.; Srivastava, S. P.; Ghosh, J. K . J. Biol. Chem. 2006, 281, 22029-38. 12. Langham, A . A.; Kaznessis, Y. N. Molecular Simulation 2006, 32, 193-201. 13. Lee, K . ; Shin, S. Y.; Kim, K . ; Lim, S. S.; Hahm, K . S.; Kim, Y . Biochem. Biophys. Res. Commun. 2004, 323, 712-9. 14. Shin, S. Y.; Hahm, K . S. Biotechnol. Lett. 2004, 26, 735-9. 15. Song, Y. M.; Yang, S. T.; Lim, S. S.; Kim, Y . ; Hahm, K. S.; Kim, J. I.; Shin, S. Y. Biochem. Biophys. Res. Commun. 2004, 314, 615-21.



62 16. Yang, J. Y.; Shin, S. Y.; L i m , S. S.; Hahm, K.-S.; K i m , Y. Journal of Microbiology and Biotechnology 2006, 16, 880-888. 17. L i m , S. S.; Song, Y . M.; Jang, M. H.; Kim, Y . ; Hahm, K . S.; Shin, S. Y . Protein and Peptide Letters 2004, 11, 35-40. 18. Shin, S. Y.; Yang, S.-T.; Eom, S. H . ; Song, W. K.; Kim, Y . ; Hahm, K.-S.; Kim, J. I. Protein and Peptide Letters 2001, 8, 281-288. 19. Khandelia, H . ; Kaznessis,Y.N.Peptides 2005, 26, 2037-49. 20. Cole, A . M.; Weis, P.; Diamond, G. J. Biol. Chem. 1997, 272, 12008-12013. 21. Andersson,M.;Boman,A.;Boman, H . G . Cell. Mol. Life Sci. 2003, 60, 599606. 22. Moore, K . S.; Bevins, C. L.; Brasseur, M. M.; Tomassini, N.; Turner, K.; Eck, H . ; Zasloff,M.J. Biol. Chem. 1991, 266, 19851-19857. 23. Sun, D.; Eccleston, E . D.; Fallon, A . M. Biochem. Biophys. Res. Commun. 1998, 249, 410-415. 24. Marchini, D.; Giordano, P. C.; Amons, R.; Bernini, L . F.; Dallai, R. Insect Biochem. 1993, 23, 591-598. 25. Bagella, L.; Scocchi, M.; Zanetti, M. FEBS Lett. 1995, 376, 225-228.


Membrane Selectivity of Antimicrobial Peptides - ACS Symposium

Recommend Documents