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Amphiphilic Peptide Interactions with Complex Biological Membranes: Effect of peptide properties on antimicrobial and anti-inflammatory effects
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. (Martin Malmsten`s group)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

With increasing problem of resistance development in bacteria against conventional antibiotics, as well as problems associated with diseases either triggered or enhanced by infection, there is an urgent need to identify new types of effective therapeutics for the treatment of infectious diseases and its consequences. Antimicrobial and anti-inflammatory peptides have attracted considerable interest as potential new antibiotics in this context. While antimicrobial function of such peptides is being increasingly understood demonstrated to be due to bacterial membrane disruption, the mechanisms of their anti-inflammatory function are poorly understood. Since bacterial membrane component lipopolysaccharide triggers inflammation, this thesis aims at clarifying importance of lipopolysaccharide (LPS)-peptide interactions while investigating possible modes of action of peptides exhibiting anti-inflammatory effect. Furthermore, effect of poly(ethylene)glycol (PEG)-conjugation was investigated to increase performance of such peptides.

Results presented in this thesis demonstrate that peptide-induced LPS- and lipid A binding/scavenging is necessary but not sufficient criterium for anti-inflammatory effects of peptides. Furthermore, preferential binding to LPS over lipid membrane, as well as higher binding affinity to the lipid A moiety within LPS, are seen for these peptides. In addition, results demonstrate that apart from direct LPS scavenging, membrane-localized peptide-induced LPS scavenging seem to contribute partially to anti-inflammatory effect. Furthermore, fragmentation and densification of LPS aggregates, in turn dependent on the peptide secondary structure on LPS binding, as well as aromatic packing interactions, correlate to the anti-inflammatory effect, thus promoting peptide-induced packing transition in LPS aggregates as key for anti-inflammatory functionality. Thus, peptide-induced LPS aggregate disruption together with reduction of the negative charge of LPS suggests the importance of phagocytosis as an alternative to the inflammatory pathway, which needs to be further investigated. Furthermore, PEG conjugation of peptide results in strongly reduced toxicity at a cost of reduced antimicrobial activity but markedly retained anti-inflammatory effect.

Taken together, the results obtained in this work have demonstrated several key issues which need to be taken into consideration in the development of effective and selective anti-inflammatory peptide therapeutics for the treatment of severe Gram-negative bacterial infections.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 216
Keyword [en]
LPS, Antimicrobial, Peptide, Inflammation, Infections, Liposome, Binding, PEG
National Category
Medical and Health Sciences
Research subject
Pharmaceutical Physical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-282781ISBN: 978-91-554-9559-6 (print)OAI: oai:DiVA.org:uu-282781DiVA: diva2:918771
Public defence
2016-06-03, B41, BMC, Husargatan 3, Uppsala, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council
Available from: 2016-05-13 Created: 2016-04-06 Last updated: 2016-06-01
List of papers
1. Membrane and lipopolysaccharide interactions of C-terminal peptides from S1 peptidases
Open this publication in new window or tab >>Membrane and lipopolysaccharide interactions of C-terminal peptides from S1 peptidases
2012 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, no 9, 2244-2251 p.Article in journal (Refereed) Published
Abstract [en]

The mechanisms underlying antimicrobial and anti-endotoxic effects were investigated for a series of structurally related peptides derived from the C-terminal region of S1 peptidases. For this purpose, results on bacterial killing were compared to those on peptide-induced liposome leakage, and to ellipsometry and dual polarization interferometry results on peptide binding to, and disordering of, supported lipid bilayers. Furthermore, the ability of these peptides to block endotoxic effects caused by bacterial lipopolysaccharide (LPS), monitored through NO production in macrophages, was compared to the binding of these peptides to LPS, and to secondary structure formation in the peptide/LPS complex. Bacteria killing, occurring through peptide-induced membrane lysis, was found to correlate with liposome rupture, and with the extent of peptide binding to the lipid membrane, no adsorption threshold for peptide insertion being observed. Membrane and LPS binding was found to depend on peptide net charge, illustrated by LPS binding increasing with increasing peptide charge, and peptides with net negative charge being unable to lyse membranes, kill bacteria, and block LPS-induced endotoxic effect. These effects were, however, also influenced by peptide hydrophobicity. LPS binding was furthermore demonstrated to be necessary, but not sufficient, for anti-endotoxic effect of these peptides. Circular dichroism spectroscopy showed that pronounced helix formation occurs in peptide/LPS complexes for all peptides displaying anti-endotoxic effect, hence potentially linked to this functionality. Similarly, ordered secondary structure formation was correlated to membrane binding, lysis, and antimicrobial activity of these peptides. Finally, preferential binding of these peptides to LPS over the lipid membrane was demonstrated.

Keyword
Antimicrobial peptide, Dual polarization interferometry, Ellipsometry, Lipopolysaccharide, Liposome, Membrane
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-180268 (URN)10.1016/j.bbamem.2012.03.017 (DOI)000306882600019 ()
Available from: 2012-09-03 Created: 2012-09-03 Last updated: 2017-12-07Bibliographically approved
2. Lipopolysaccharide Interactions of C-Terminal Peptides from Human Thrombin
Open this publication in new window or tab >>Lipopolysaccharide Interactions of C-Terminal Peptides from Human Thrombin
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2013 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, no 5, 1482-1492 p.Article in journal (Refereed) Published
Abstract [en]

Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRL-KKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-202380 (URN)10.1021/bm400150c (DOI)000319034600027 ()
Available from: 2013-06-24 Created: 2013-06-24 Last updated: 2017-12-06Bibliographically approved
3. Effects of linear amphiphilicity on membrane interactions of C-terminal thrombin peptides
Open this publication in new window or tab >>Effects of linear amphiphilicity on membrane interactions of C-terminal thrombin peptides
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2014 (English)In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 4, no 71, 37582-37591 p.Article in journal (Refereed) Published
Abstract [en]

Effects of linear amphiphilicity on membrane interactions of antimicrobial peptides were investigated by ellipsometry, dual polarization interferometry, fluorescence spectroscopy, light scattering, and circular dichroism. In doing so, the thrombin-derived GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) was compared to WFF25 (WFFFYYLIIGGGVVTHQQRKKKKDE) of identical composition, but with amino acids sorted according to hydrophobicity, the latter peptide thus displaying pronounced linear amphiphilicity. In addition, GKY25d (GKYG(f) YTH(v) FRL(k) KWI(q) KVI(d) QFGE; with an identical sequence but with selected D-amino acid substitutions) was included as a control peptide, for which conformationally induced (helix-related) amphiphilicity was suppressed. Through its pronounced linear amphiphilicity, WFF25, but not the less amphiphilic GKY25 and GKY25d, forms aggregates in solution. Through its terminal W/F stretch, WFF25 also displays pronounced selectivity, with higher membrane binding and liposome rupture than GKY25 and GKY25d for anionic membranes, but suppressed peptide insertion and lytic effects for zwitterionic ones. In addition, WFF25 binds extensively to anionic polyelectrolyte components in bacterial membranes, i.e., lipopolysaccharide and lipoteichoic acid, resulting in reduced antimicrobial effects through peptide scavenging, not seen for the less amphiphilic GKY25 and GKY25d peptides. Taken together, the results thus demonstrate a series of striking effects for highly amphiphilic peptides, which need to be recognized in the development of such compounds as potential peptide therapeutics.

National Category
Clinical Medicine Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-233621 (URN)10.1039/c4ra05420b (DOI)000341454600018 ()
Available from: 2014-10-07 Created: 2014-10-07 Last updated: 2017-12-05Bibliographically approved
4. Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides
Open this publication in new window or tab >>Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides
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2013 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 11, 2709-2719 p.Article in journal (Refereed) Published
Abstract [en]

Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLERKLTHRLFRRNEGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure-activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics. 

Keyword
Antimicrobial peptide, Dual polarization interferometry, Ellipsometry, Lipopolysaccharide, Liposome, Membrane
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-211434 (URN)10.1016/j.bbamem.2013.06.015 (DOI)000326143200038 ()
Available from: 2013-11-27 Created: 2013-11-25 Last updated: 2017-12-06Bibliographically approved
5. Effects of PEGylation on Membrane and Lipopolysaccharide Interactions of Host Defense Peptides
Open this publication in new window or tab >>Effects of PEGylation on Membrane and Lipopolysaccharide Interactions of Host Defense Peptides
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2014 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, no 4, 1337-1345 p.Article in journal (Refereed) Published
Abstract [en]

Effects of poly(ethylene glycol) (PEG) conjugation on peptide interactions with lipid membranes and lipopolysaccharide (LPS) were investigated for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), an antimicrobial and anti-inflammatory peptide derived from human heparin cofactor II. In particular, effects of PEG length and localization was investigated by ellipsometry, circular dichroism, nanoparticle tracking analysis, and fluorescence/electron microscopy. PEGylation of KYE28 reduces peptide binding to lipid membranes, an effect accentuated at increasing PEG length, but less sensitive to conjugation site. The reduced binding causes suppressed liposome leakage induction, as well as bacterial lysis. As a result of this, the antimicrobial effects of KYE28 is partially lost with increasing PEG length, but hemolysis also strongly suppressed and selecticity improved. Through this, conditions can be found, at which the PEGylated peptide displays simultaneously efficient antimicrobial affects and low hemolysis in blood. Importantly, PEGylation does not markedly affect the anti-inflammatory effects of KYE28. The combination of reduced toxicity, increased selectivity, and retained anti-inflammatory effect after PEGylation, as well as reduced scavenging by serum proteins, thus shows that PEG conjugation may offer opportunities in the development of effective and selective anti-inflammatory peptides.

National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-225069 (URN)10.1021/bm401884e (DOI)000334571600026 ()
Available from: 2014-06-23 Created: 2014-05-27 Last updated: 2017-12-05Bibliographically approved
6. Role of Aromatic Amino Acids in Lipopolysaccharide and Membrane Interactions of Antimicrobial Peptides for use in Plant Disease Control
Open this publication in new window or tab >>Role of Aromatic Amino Acids in Lipopolysaccharide and Membrane Interactions of Antimicrobial Peptides for use in Plant Disease Control
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2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 25, 13301-13317 p.Article in journal, Meeting abstract (Refereed) Published
Abstract [en]

KYE28(KYEITTIHNLFRKLTHRLFRRNFGYTLR), the representative sequence  of helix D of heparin co-factor II, was demonstrated to be potent against agronomically important Gram-negative plant pathogens X. vesicatoria and X. oryzae,capable of inhibiting disease symptoms in detached tomato leaves. NMR studies in presence of lipopolysaccharide provided structural insights into the mechanisms underlying this, notably in relation to outer membrane permeabilisation. The three-dimensional solution structure of KYE28 in LPS is characterised by a N-ter helical segment, an intermediate loop and an extended C-ter. The two termini are in close proximity to each other via aromatic packing interactions, while the positively charged residues formed an exterior polar shell. To further demonstrate the importance of the aromatic residues for this, a mutant peptide KYE28A, with Ala substitutions at F11, F19, F23 and Y25 showed attenuated antimicrobial activity at high salt concentrations, as well as lower membrane disruption and LPS binding abilities compared to KYE28. In contrast to KYE28, KYE28A adopted an opened out helical structure in LPS with extended N- and C-ter and a small break in between the helical segments. Aromatic packing interactions were completely lost, although hydrophobic interaction between the side chains of hydrophobic residues were still partly retained, imparting an amphipathic character and explaining its residual antimicrobial activity and LPS binding as observed from ellipsometry and ITC. We thus present important structural aspects of KYE28, constituting an aromatic zipper, of potential importance, for the development of novel plant protection agents and therapeutic agents.

Keyword
LPS, Antimicrobial
National Category
Medical and Health Sciences
Research subject
Pharmaceutical Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-282778 (URN)10.1074/jbc.M116.719575 (DOI)000379770500033 ()27137928 (PubMedID)
Available from: 2016-04-06 Created: 2016-04-06 Last updated: 2017-11-30Bibliographically approved

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