Hein-Kristensen, Line

Antibiotika har været brugt til behandling af bakterieinfektioner gennem mere end 50 år. Bakterieresistens overfor konventionelle antibiotika er dog steget drastisk de seneste årtier, hvilket begrænser antallet af behandlingsmuligheder mod komplicerede bakterieinfektioner. I jagten på alternative antibakterielle stoffer har de antimikrobielle peptider (AMPer) fået meget opmærksomhed eftersom deres aktivitet er rettet mod bakteriernes akilleshæl dvs. deres særlige membranstruktur. Disse forsvarsstoffer findes overalt i det naturlige miljø, da de udgør en del af immunforsvaret hos alle livsformer. Adskillige af disse stoffer er derfor blevet karakteriseret og udviklet som fremtidens antibiotika. Derudover er syntetiske analoger, peptidomimetics, blevet designet ud fra strukturen af naturlige AMPer, hvilket giver mulighed for at forbedre den antibakterielle aktivitet.Formålet med dette ph.d. projekt var at etablere en sammenhæng mellem struktur og antibakteriel aktivitet i en række a-peptid/ß-peptoid peptidomimetics, og derudover at klarlægge hvilke mekanismer, der kunne forklare eventuelle forskelle i aktivitet. Vi bestemte aktiviteten af disse peptidomimetics mod en række fødevarebårne og hospitals-erhvervede patogene bakterier. Dette studie af forholdet mellem struktur og aktivitet viste, at en vis peptid-længde var nødvendig for at opnå høj antibakteriel aktivitet, da analoger med en længde på mindre end 12 enheder var næsten ineffektive. I det indeværende design, hvor der er en 1:1 ratio mellem de kationiske a-aminosyrer og hydrofobiske ß-peptoider, havde aminosyre-komposition og chiralitet i ß-peptoid enheden kun ringe indflydelse på den antibakterielle aktivitet.Vi brugte et ATP lækage assay til at vise, at den antibakterielle aktivitet skyldtes permeabilisering eller ødelæggelse af bakteriens cellemembran. Dette forårsagede ændringer af celleoverfladen, som blev visualiseret gennem Skanning Elektron Mikroskopi (SEM). Det er vigtigt at understrege, at lækage studierne blev lavet med levende bakterier og at vi brugte en koncentration, som var tæt på den Minimum Inhibitoriske Koncentration (MIC). Vores undersøgelser viste, at alle analogerne har den samme virkningsmekanisme og at denne er uafhængig af bakterien. Resultaterne viste dog også, at den præcise interaktion med membranen kan variere eftersom der var store variationer i mængden af lækket ATP og det efterfølgende fald i antallet af levende celler. En serie på tre peptider, som kun varierede i længden, forårsagede alle ATP lækage, men kun den længste bevirkede, at cellen blev tømt for ATP. Dette korrelerede med et markant fald i antallet af levende celler.Kontinuerlig selektion gennem 500 generationer resulterede i, at 10 ud af 10 linjer af Escherichia coli udviklede resistens mod den benyttede analog. Dette var det første bevis på udvikling af resistens mod peptidomimetics, selvom adskillige studier har vist, at AMP resistens kan udvikles. Vi anser resistensen for at være specifik for analogerne i indeværende peptid-bibliotek, da vi ikke fandt tilfælde af kryds-resistens til en række andre AMPer. Vi sekventerede hele genomet fra seks højresistente isolater fra to forskellige linjer, og identificerede en enkeltmutation (SNP) i genet, der koder for MltD proteinet. Dette protein er involveret i reorganiseringen af peptidoglycan laget og eftersom denne mutation kun forekom i isolater, som var meget resistente, anser vi det for sandsynligt, at en ændring i dette protein er årsagen til resistens.Disse resistente isolater havde forøget sensitivitet overfor humant blodplasma, hvilket kunne skyldes tilstedeværelsen af innate effektormolekyler. Tilsætning af 50 % blodplasma forøgede samtidig aktiviteten af analogerne op til 32 gange. Denne effekt forsvandt efter varme-behandling, som er en metode, der benyttes til at inaktivere komplementsystemet.Arbejdet i denne afhandling har medvirket til en øget forståelse for hvordan centrale strukturelle egenskaber påvirker den antibakterielle aktivitet. Peptidomimetics kan betragtes som et godt bud på fremtidens antibiotika, fordi det er muligt at optimere deres egenskaber gennem ændringer i deres struktur, hvilket betyder uanede muligheder for variation. Denne afhandling konkluderer, at den antibakterielle aktivitet kan forbedres yderligere, og at fremtidig resistens måske kan afværges ved optimering af den nuværende design.Antibiotics have been an effective weapon against bacterial infections for over 50 years. However, bacterial resistance towards conventional antibiotics has increased considerably within the last decades and the number of antibacterial agents available for treating complicated bacterial infections is becoming increasingly limited. In the search for alternatives therapies, antimicrobial peptides (AMPs) have received considerable attention since they target the bacterial Achilles’ heel i.e. their distinct membrane structure. These host defence molecules are ubiquitous in nature by forming part of the immune system among all classes of life. Several of these compounds have therefore been characterised and developed into future antibacterials. Furthermore, in an attempt to improve the antibacterial activity, synthetic analogues i.e. peptidomimetics have been designed based on the structural properties of natural AMPs.The purpose of this PhD study was to establish the potential correlation between structure and antibacterial activity for a series of a-peptides/ß-peptoid peptidomimetics and additionally to determine if mechanistic differences could explain observed variations in activity. We determined the activity of the peptidomimetics against a range food borne and nosocomial pathogenic bacteria. These structure-activity studies demonstrated that peptide length was important for high antibacterial activity since analogues with a length shorter than 12 residues were virtually inactive. In the present design, with a 1:1 ratio between cationic a-amino acids and hydrophobic ß-peptoids, amino acid composition and chirality in the ß-peptoid unit only had a minor influence on antibacterial activity.By using an ATP leakage assay we determined that the mechanism of action of the chimeras was permeabilization or disruption of the bacterial cell membrane. The resulting changes to the cell surface were visualised with Scanning Electron Microscopy (SEM). Importantly, our leakage studies were performed with viable bacterial cells and using a concentration that was close to the Minimum Inhibitory Concentration (MIC). The findings show that all of the chimeras included in the study have a similar mechanism of action that was independent on bacterial species. However, the study showed that the detailed interaction with the cell membrane may be different, since there were large variations in the amount of leaked ATP and subsequent loss of viability. A series of three peptides differing only in length all caused ATP leakage but only the longest of the three caused complete depletion of intracellular ATP, which correlated with a substantial loss in the number of viable cells.In a continuous selection protocol encompassing 500 generations, 10 out of 10 lineages of Escherichia coli developed resistance towards the chimera they had been exposed to. This was the first time resistance was successfully developed towards peptidomimetics, though several studies have reported resistance towards AMPs. Resistance was specific to compounds within the peptidomimetics library, since we were unable to demonstrate cross-resistance to other AMPs. We sequenced the entire genome of six highly resistant isolates from two separate lineages, and identified a single-nucleotide-polymorphism (SNP) in the gene encoding the MltD protein. This protein functions in the reorganization of the peptidoglycan layer, and we consider it likely that a change in this protein is the cause of resistance, since the SNP was found exclusively in isolates with high levels of resistance.Conversely, these resistant isolates displayed increased sensitivity towards human blood plasma possibly due to immune effector compounds present in this. The addition of 50 % blood plasma also increased the activity of the chimeras against wild type bacteria by up to 32 times. This effect was abolished by heat-treatment, which is a method known to inactivate the complement system.The findings in this thesis have elucidated how central structural determinants influence antibacterial activity. Peptidomimetics can be regarded as promising future antibiotics since the possibility to optimize their properties through structural modification allows for continuous variation. This thesis concludes that antibacterial activity can be improved further and that in the future resistance may be circumvented by optimizing the existing scaffold

Online Research Database In Technology

Spectrum and activity of novel antimicrobial peptidomimetics

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Spectrum and activity of novel antimicrobial peptidomimetics

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