Characterization of FimH in Escherichia coli Isolated from Ulcerative Colitis Patients and Healthy Controls -Inhibition of bacterial adhesion by anti-fimH monoclonal antibodies

Colitis ulcerosa (UC) er en undergruppe af sygdommen inflammatorisk tarmsygdom (IBD). Der er en multifaktoriel gruppe af gastro-intestinale lidelser hvis etologi endnu ikke er fuldt afdækket. Patienter der lider af IBD har oftest en øget forekomst af Escherichia coli (E. coli) i deres tarm mikrobiota sammenlignet med raske personer. Hvilket indikerer en mulig sammenhæng mellem disse E. coli og sygdommen. Dette speciale havde derfor til mål at karakterisere adhæsionet fra type 1 fimbrier (FimH) fra E. coli isoleret fra UC-patienter og raske personer, samt undersøge effekten af anti-FimH monoklonale antistoffer på stammernes adhæsion. Til det blev der anvendt en række metoder; fimH sekvenstypning, transformation med plasmidet pPKL91, der indeholder fimbrial regulatoren fimB, vurdering af udtrykket af type 1 fimbrier ved hjælp af en fim-Switch test, adhæsion til mannan og RNaseB, indirekte ELISA for at differencerne epitoper, agglutination til røde blodlegemer og gær, samt adhæsion til epitelceller og alternative kvantificeringsmetoder som flow cytometri og RT-qPCR.Resultaterne af disse forsøg afslørede flere indsigter i FimH-varianterne hos patienter med UC. Patienter med UC viste en højere forekomst af mutationer i fimH-genet sammenlignet med raske kontrolpersoner, hvilket antyder en potentiel sammenhæng mellem fimH og udvikling af UC. FimH-profilerne viste en større lighed mellem FimH-varianterne hos patienter med UC og dem, der normalt associeres med urinvejsinfektioner (UTI), snarere end de fækale E. coli-stammer, der findes hos raske personer. FimH-varianterne kunne ikke alene forklare virkningen af de anti-FimH-monoklonale antistoffer. Antistoffet mAb824 viste evnen til at hæmme adhæsionen af både kontrol- og UC -stammer lige effektivt, hvilket antyder dens potentielle effektivitet mod mange forskellige FimH-varianter. Dette speciale kan gennem karakteriseringen af FimH-varianter hos patienter med UC og deres potentielle indflydelse på effektiviteten af anti-FimH-monoklonale antistoffer bidrage til forståelsen af UC’s patogenese og vise en vej for en mulig behandling af denne sygdom. Dog er yderligere forskning afgørende for at afdække de præcise mekanismerne bag de iagttagelser beskrevet i dette speciale.Ulcerative colitis (UC) is a subtype of inflammatory Bowel Disease (IBD) which is a complex and multifactorial group of gastrointestinal disorders. Although the etiology of IBD is still not completely understood, patients suffering from IBD often exhibit an increased prevalence of Escherichia coli (E. coli) within their gut microbiota, compared to healthy individuals. Which suggests that there is a potential link between this bacterium and the disease. This thesis aimed to investigate this connection by characterizing the adhesin of type 1 fimbriae (FimH) from E. coli strains isolated from fecal samples of UC patients and healthy controls as well as the effect of anti-FimH antibodies on these strains. Various assays were employed; fimH sequence typing, transformation with plasmid pPKL91 harboring the fimbrial regulator fimB, assessment of the expression of the type 1 fimbriae by a fim-Switch assay, adhesion to mannan and RNaseB, indirect ELISA to differentiate mAb824 and mAb320, agglutination to red blood cells and yeast and lastly adhesion to epithelial cells and alternative quantification methods such as flow cytometry and RT-qPCR.The findings of this thesis revealed several insights into FimH variations in UC patients. FimH originated from UC patients exhibited a higher incidence of mutations in the fimH gene compared to the healthy controls, suggesting a potential association between fimH and the development or progression of UC. Furthermore, the fimH profiles from UC patients demonstrated a closer resemblance to those typically associated with urinary tract infection (UTI) strains, rather than the fecal E. coli found in healthy individuals. Notably, the FimH variations alone could not entirely account for the effect of anti-FimH monoclonal antibodies. The antibody mAb824 exhibited the ability to inhibit adhesion in both control and UC strains equally. This suggests that this antibody have a potential effectiveness against a wide range of FimH variants. In conclusion, this thesis has elucidated the existence of FimH variations in UC patients and controls and the effect of anti-FimH monoclonal antibodies. These findings can contribute to the understanding of UC pathogenesis and provide a potential path for therapeutic interventions in this disease. Nonetheless, additional research is essential to validate and find the precise mechanisms underlying these observations.<br/