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dc.contributor.advisorHaug, Tor
dc.contributor.authorDey, Hymonti
dc.date.accessioned2024-05-24T10:37:29Z
dc.date.available2024-05-24T10:37:29Z
dc.date.issued2024-06-06
dc.description.abstract<p>Modern medicine is threatened by the emergence of antimicrobial resistance (AMR). New agents with unique structures and mechanisms of action are urgently needed to address the problems associated with AMR. To effectively address this challenge, a proper understanding of the structure-activity relationship is required to decipher the mechanism of action of novel compounds. Template-based design inspired by the structural motif of natural antimicrobial peptides (AMPs) could open new ways to develop antimicrobial agents with novel mechanisms of action. In this thesis, the biological effects of altering the amino acid composition, cyclization, and addition of N-terminal fatty acid chains on short synthetic marine AMP derivatives were studied. A rational peptide design approach based on well-known physicochemical features, such as hydrophobicity and cationic properties was applied on templates derived from the marine AMPs Turgencin A and EeCentrocin 1. Antimicrobial screening was conducted using different Gram-positive and Gram-negative bacterial strains as well as different fungal strains. Several bacterial biosensors including novel MoA-specific whole-cell biosensors were used to study the mechanism of action (MoA). <p>The incorporation of tryptophan into the core sequence of Turgencin A analogues as well as the substitution of lysine with arginine and cyclization via disulfide bridges, increased the potency of the derivates. This indicates the importance of arginine and tryptophan residues in the improvement of amphipathic properties. The highest antimicrobial activity was observed for cyclic lipopeptides C12-cTurg-1 and C8-cTurg-2, indicating the importance of acylation in enhancing both activity and stability via increased hydrophobicity and cyclization. Similarly, modification with arginine and tryptophan or head-to-tail cyclization of a previously reported lead peptide (P6) derived from the heavy chain of the marine heterodimeric peptide EeCentrocin 1 resulted in the potent derivatives P6-W6R8 and cP6-W6R8. Structure-activity studies showed that derivatives with increased antibacterial and membranolytic activities and low toxicity can be generated by fine-tuning several modifications. In addition to their effects on the cytoplasmic membrane, a secondary mechanism, involving outer membrane (OM) disruption in Gram-negative bacteria and the induction of stress responses related to membrane damage, suggests a concentration-dependent mechanism of action. In subsequent MoA studies, the application of a novel bacterial biosensor for measuring OM activity confirmed the primary interaction of the synthetic derivatives with the OM of <i>Escherichia coli</i>. <p>In studies involving combined treatment with either erythromycin or vancomycin, synergy against <i>E. coli</i> occurred more frequently with the OM active peptides, whereas no synergy was observed against Gram-positive bacteria. Most analogues were able to inhibit the formation of both Gram-positive and Gram-negative bacterial biofilms, which may be attributed to their membranolytic and killing effect. However, the biofilm inhibitory effects of moderately active analogues were independent of their growth inhibition effects. Synergistic combinations were also able to inhibit biofilm formation by <i>Salmonella enterica</i> serovar Typhimurium UMR1 and <i>Pseudomonas aeruginosa</i> PA01. The findings of the present study support previous research which shows that OM-acting AMPs potentiates the effects of antibiotics, by facilitating their access to the intracellular target molecules. <p>In summary, this thesis demonstrates that the targeted modifications of the structural motifs of marine AMPs can improve their antimicrobial and membranolytic activities, as well as their potential as synergists - to potentiate the antimicrobial and antibiofilm effects of commercial antibiotics.en_US
dc.description.abstract<p>Moderne medisin er truet av fremveksten av antimikrobiell resistens (AMR). Det er derfor et behov for nye legemidler med unike strukturer og virkningsmekanismer for å løse problemene knyttet til AMR. Den kjemiske strukturen til naturlige antimikrobielle peptider (AMP) kan være til inspirasjon og fungere som modeller til å utvikle nye antimikrobielle midler med nye virkningsmekanismer. Lav selektivitet og moderat antimikrobiell aktivitet utgjør en utfordring for utviklingen av AMP som antibiotika. For å håndtere denne utfordringen, kreves det god forståelse av struktur-aktivitet-forholdet for å bestemme virkningsmekanismen til disse nye forbindelsene. En rasjonell peptid-design tilnærming, basert på velkjente fysisk-kjemiske egenskaper, som hydrofobisitet og kationiske egenskaper, ble benyttet på templater avledet fra de marine AMP-ene Turgencin A og EeCentrocin 1. I denne avhandlingen ble de biologiske effektene av endret aminosyresammensetning, syklisering, og påkobling av N-terminale fettsyrekjeder på syntetiske AMP-derivater studert. Studier av antimikrobiell aktivitet og virkningsmekanismer ble utført ved bruk av ulike Gram-positive og Gram-negative bakteriestammer, samt virkningsmekanisme-spesifikke biosensorstammer. <p>Inkorporering av tryptofan i den sentrale PGG-sekvensen til Turgencin-derivater samt substitusjon av lysin med arginin og syklisering via disulfidbroer, økte aktiviteten til derivatene. Dette viser betydningen av aminosyrene arginin og tryptofan i forbedringen av peptidenes amfipatiske egenskaper. Den høyeste antimikrobielle aktiviteten ble imidlertid oppnådd for de sykliske lipopeptidene C12-cTurg-1 og C8-cTurg-2, noe som indikerer viktigheten av acylering som en metode for å forbedre hydrofobisitet og syklisering for å forbedre aktivitet og stabilitet. Inkorporering med arginin og tryptofan, samt «hode-til-hale»-syklisering av et tidligere rapportert peptid (P6, avledet fra den tunge kjeden til det marine heterodimere peptidet EeCentrocin 1), resulterte i de potente derivatene P6-W6R8 og cP6-W6R8. Struktur-aktivitets-studier indikerer at derivater med økt antibakteriell og membranolytisk aktivitet og lav toksisitet kan skapes ved å finjustere flere modifikasjoner. I tillegg til deres effekter på bakterienes cytoplasmamembran, antydes også sekundære mekanismer som involverer ødeleggelse av bakterienes yttermembran og induksjon av cellevegg-mediert stressrespons, med andre ord en konsentrasjonsavhengig dobbel virkningsmekanisme. Videre virkemekanisme-studier med bruk av en ny bakteriell biosensor for måling av ytre membranaktivitet, bekreftet den primære interaksjonen mellom de syntetiske derivatene og yttermembranen til den Gram-negative bakterien <i>Escherichia coli</i>. <p>Ved å kombinere de syntetiske peptidene med antibiotikaene erytromycin eller vankomycin forekom det synergi mot <i>E. coli</i>, men ikke mot Gram-positive bakteriestammer. De fleste peptidanalogene var imidlertid i stand til å hemme dannelsen av biofilm fra både Gram-positive og Gram-negative bakterier, noe som kan skyldes deres membranolytiske og drepende effekt. Biofilmhemmende effekter av moderat aktive analoger var imidlertid uavhengig av deres vekstinhibering. Synergistiske kombinasjoner var også i stand til å hemme biofilmdannelse av de Gram-negative stammene <i>Salmonella enterica</i> serovar Typhimurium UMR1 og <i>Pseudomonas aeruginosa</i> PA01. Funnene fra dette studiet støtter tidligere forskning som viser at yttermembran-virkende AMP forsterker effekten av antibiotika, bl.a. ved å lette deres tilgang til de intracellulære målmolekylene. <p>Oppsummert viser denne avhandlingen at modifikasjoner av de strukturelle motivene til marine AMP kan forbedre deres antimikrobielle og membranolytiske aktiviteter, samt øke deres potensiale som synergister - ved å forsterke kommersielle antibiotika sine antimikrobielle og antibiofilm-effekter.en_US
dc.description.doctoraltypeph.d.en_US
dc.description.popularabstractIn this thesis, two series of short synthetic peptide analogues were investigated for their antimicrobial activities against a panel of Gram-positive and Gram-negative bacterial and fungal strains. The peptides series developed are the results of structural modifications of lead peptides derived from the marine antimicrobial peptides (AMPs) Turgencin A and EeCentrocin 1. The modifications included amino acid substitutions, cyclization, and fatty acid conjugation. All these modifications were shown to increase the antimicrobial activities. The use of mode-of-action specific biosensor strains revealed both plasma membrane and outer membrane disruptive effects for selected peptides. The peptides were shown to interfere with bacterial membrane integrity and thereby kill the bacteria in a matter of minutes. Additionally, the study revealed that certain analogues demonstrated synergistic actions when combined with antibiotics like erythromycin and vancomycin. Also, some peptides showed promising results regarding inhibition of formation and eradication of bacterial biofilms. These findings not only shed light on the importance of specific structural modifications of peptides but also hint at their use as a potential strategy to overcome the challenges regarding antimicrobial resistance.en_US
dc.description.sponsorshipThis PhD was a part of Lead-ScAMR project with financial support from UiT – The Arctic University of Norway, Tromsø.en_US
dc.identifier.isbn978-82-8266-260-4
dc.identifier.urihttps://hdl.handle.net/10037/33605
dc.language.isoengen_US
dc.publisherUiT The Arctic University of Norwayen_US
dc.publisherUiT Norges arktiske universiteten_US
dc.relation.haspart<p>Paper I: Dey, H., Simonovic, D., Hagen, I.N.-S., Vasskog, T., Fredheim, E.G.A., Blencke, H.M., … Haug, T. (2022). Synthesis and Antimicrobial Activity of Short Analogues of the Marine Antimicrobial Peptide Turgencin A: Effects of SAR Optimizations, Cys-Cys Cyclization and Lipopeptide Modifications. <i>International journal of molecular sciences, 23</i>(22), 13844. Also available in Munin at <a href=https://hdl.handle.net/10037/27420>https://hdl.handle.net/10037/27420</a>. <p>Paper II: Simonovic, D., Dey, H., Johansen, N., Anderssen, T., Hansen, I.K.Ø., Devold, H., Vasskog, T., … Strøm, M.B. Antimicrobial activity of short analogues of the marine peptide EeCentrocin 1: Synthesis of lipopeptides and head-to-tail cyclic peptides and mechanism of action studies. (Manuscript). <p>Paper III: Richard, C.S.M., Dey, H., Øyen, F., Maqsood, M. & Blencke, H.M. (2023). Outer Membrane Integrity-Dependent Fluorescence of the Japanese Eel UnaG Protein in Live <i>Escherichia coli</i> Cells. <i>Biosensors, 13</i>(2), 232. Also available in Munin at <a href=https://hdl.handle.net/10037/30492>https://hdl.handle.net/10037/30492</a>. <p>Paper IV: Dey, H., Simonovic, D., Richard, C.S.M., Hagen, I.N.-S., Johansen, N., Fredheim, E.G.A., … Blencke, H.M. Combining outer membrane active synthetic antimicrobial peptides with vancomycin or erythromycin increases antibacterial and antibiofilm activities. (Manuscript).en_US
dc.rights.accessRightsopenAccessen_US
dc.rights.holderCopyright 2024 The Author(s)
dc.subject.courseIDDOKTOR-002
dc.subjectMarine antimicrobial peptidesen_US
dc.titleAntimicrobial activities and mechanisms of action of peptide analogues of marine originen_US
dc.typeDoctoral thesisen_US
dc.typeDoktorgradsavhandlingen_US


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