Madsen Sommer, Lea Mette

I århundreder har man undersøgt evolution ved at bruge en "endpoint" fremgangsmåde. Det vil sige, at man primært har lavet sammenligninger imellem arter og ved lejlighed fossiler. Men for at kunneforstå processer generelt, inklusiv evolution, er det uvurderligt at kunne undersøge processen undervejs og derved observere den "live".Dette er muligt gennem laboratorieeksperimenter på grund af den høje grad af kontrol og klarhed. Men for at få en sand forståelse af evolution og de komplekse mekanismer det indebærer, er det nødvendigt at kombinere laboratorieforsøg med undersøgelser af naturligemiljøer.På grund af heterogeneciteten og den konstante forandring af naturligemiljøer er den primære forhindring i dette tilfælde observationen af den samme population over tid, specielt hvis populationen er lille.Ikke desto mindre har det vist sig at være muligt: Kroniske luftvejsinfektioner hos cystisk fibrose (CF) patienter har vist sig at give etenestående indblik i adaptation og evolution af Pseudomonas aeruginosa til dette naturlige miljø. Det har tilmed været muligt at følge denne proces over tusinder af bakterielle generationer. På grund af den langvarige og persisterende kolonisering og infektion af disse patienter, er CF infektionssystemet en værdifuld model til undersøgelser af evolutionære processer. Det primære fokus for denne afhandling har været at vise links imellemevolutionære studier fra CF modelsystemer og generelle evolutionsteorier, hvoraf de fleste oprindeligt blev udviklet via observationer fra andre organismer.Denne sammenligning blev i første omgang forsøgt ved at påvise muligheden for at bruge omfattende longitudinelle isolat samlinger til studier af evolution (Studie 1). Dette blev opnået ved sammenligningaf fem sputummetagenomer med longitudinelle enkelt isolater fra fire CF patienter og identifikationen af klare genetiske links indenfor den enkelte patients specifikke P. aeruginosa population. Dette genetiskelink mellem enkelte isolater og metagenomer blev tilmed påvist for to populationer, hvor en umiddelbar patient-til-patient transmissionhavde foregået og derved øget ligheden imellem de to populationer signifikant, i forhold til populationerne fra de andre patienter.ixEfterfølgende blev en omfattende samling af 474 longitudinelle P. aeruginosa isolater udsat for hel-genom sekvensering (Studie 2). Disse isolater stammede fra 34 unge Danske CF patienter. Denne samlingblev analyseret for at rekonstruere den umiddelbare evolutionære historie og identificere gener, der var vigtige for den indledende adaptationtil CF luftveje. Fra denne analyse identificerede vi fælles klontyper mellem patienter, beviser for patient-til-patient transmission,historiske sammenhænge mellem mutationer og konvergerende evolution af 52 kandidat pathoadaptive gener.Yderligere sekvensering, af 26 P. aeruginosa isolater fra fire Italienske CF patienter (Studie 3) og 35 P. aeruginosa isolater fra 12 primær ciliedyskinesi (PCD) patienter (Studie 4), gjorde os i stand til at finde genetiske og fænotypiske links på tværs af landegrænser og sygdomme.Alle tre studier (ikke inkluderende metagenom studiet) havde fælles klontyper og klare overlap af adaptive mutationer. Men det genetiskeoverlap imellem CF og PCD isolater kunne ikke udvides til at inkludere fænotypiske overlapninger, hvilket indikerer, at det slim,der findes i luftvejene, og som er markant anderledes for CF patienter,er en signifikant selektionsfaktor for evolutionen og adaptationen af P. aeruginosa i disse miljøer.Uafhængigt af hinanden, men også i kombination giver de studier præsenteret i denne afhandling ny og udvidet viden omkring adaptation og evolution til både CF og PCD luftveje. Ved yderligere karakterisering af genetiske og fænotypiske adaptationer burde det væremuligt at oversætte disse resultater til klinisk relevant information,der kan lede til vigtig viden med hensyn til behandlingsstrategier og muligvis også ekstrapolation til andre infektionsscenarier. OVERORDNET: Igennem den konvergerende genetiske og fænotypiske adaptation, som er observeret i CF studier og ved at linke dette til evolutionære processer viser denne afhandling at samlinger af longitudinelle P. aeruginosa isolater fra CF patienter giver en værdifuldbasis for studier af adaptation og evolution i naturlige miljøer.For centuries evolution has been investigated with an "end-point" approach,through comparisons between species or fossil records. However,to understand processes in general, including evolution, it is highly valuable to observe the dynamics as they unfold, in "realtime".This is possible through laboratory experiments, with a high degree of control and rigour. But to truly understand evolution and the complex mechanisms it deploys, it is necessary to combine the laboratory learnings with investigations of natural systems. –Though, this can be tricky. Because of the heterogeneity and constant change of natural environments, the primary obstacle is re-sampling of the samepopulation over time, especially if the population is small.Nevertheless, it has been accomplished: Chronic airway infections of cystic fibrosis (CF) patients have offered a unique view into the adaptationand evolution of Pseudomonas aeruginosa to this natural environment,spanning thousands of bacterial generations. Because of the prolonged and persistent infections, they provide a valuable modelsystem for the investigation of evolutionary mechanisms.The main focus of this thesis has been to show the link between evolutionary studies in the CF model system and general evolutionary theories, many of which have been developed from observations of other organisms.This comparison has initially been sought by showing the plausibility of using comprehensive collections of longitudinally sampled single isolates, for their use in evolutionary studies (Study 1). This was done by comparing five metagenomes with single isolates from four CF patients, and identifying significant genetic links found within the patient specific P. aeruginosa populations. This evident genetic link was even found for two populations, where a recent patient-to-patienttransmission had occurred.Secondly a comprehensive collection of 474 longitudinal single P. aeruginosa isolates from 34 young Danish CF patients was investigatedby whole genome sequencing (Study 2). This was done to reconstruct the recent evolutionary history, and identify genes targeted in the initial adaptation to the CF airways. From this analysis we found common clonal lineages among the patients, evidence of patient-to-patient transmission, historic contingencies, and convergent evolution of 52 candidate pathoadaptive genes. By further genome sequencing 26 P. aeruginosa isolates from four Italian CF patients (Study 3), and 35 P. aeruginosa isolates from 12 primary ciliary dyskinesia (PCD) patients (Study 4), we were able to find genetic and phenotypic links across countries and diseases.All three studies (not including the metagenome study) had common clonal lineages and clear overlaps of genetic adaptational patterns.However, the genetic overlap between CF and PCD isolates did not extend to a phenotypic overlap, which indicates that the mucus, which is different in CF patients compared to PCD patients, is a significant selective factor for the evolution and adaptation of P. aeruginosa to these environments.Independently and together the studies presented in this thesis provide new knowledge of adaptation and evolution in both CF and PCD airways. With further characterisation of genetic and phenotypic adaptationsit should be possible to translate these results into clinically relevant information, leading to better epidemiological predictions,valuable information with regards to treatment strategies, and perhaps extrapolation of this knowledge to other infection scenarios. OVER ALL: Through the convergence of genetic and phenotypic adaptations observed in CF studies and by linking processes of evolution to these observations, this thesis shows that collections of longitudinal P. aeruginosa isolates from CF patients provide a valuable basis for the study of adaptation and evolution in natural environments

Online Research Database In Technology

Evolution and adaptation of <i>Pseudomonas aeruginosa</i> in cystic fibrosis airways:cystic fibrosis as a model system

http://orbit.dtu.dk/ws/files/124303721/Evolution_and_adaptation_of_Pseudomonas_aeruginosa_in_cystic_fibrosis_airways.pdf

Evolution and adaptation of <i>Pseudomonas aeruginosa</i> in cystic fibrosis airways:cystic fibrosis as a model system

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