Title UNUTARVAKUOLARNI ŽIVOT FRANCISELLA NOVICIDA U DICTYOSTELIUM DISCOIDEUM Title (english) INTRAVACUOLAR LIFE CYCLE OF FRANCISELLA NOVICIDA IN DICTYOSTELIUM DISCOIDEUM Author Valentina Marečić Mentor Marina Šantić (mentor)Committee member Darinka Vučković (predsjednik povjerenstva)Committee member Ana Budimir (član povjerenstva)Committee member Vladimir Mićović (član povjerenstva)Committee member Marina Šantić (član povjerenstva)Granter University of Rijeka Defense date and country 2019, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 616 - Pathology. Clinical medicine ThesaurusMESH - Medical Subject Headings )
Francisella
Tularemia
Phagosomes
Dictyostelium
Vacuoles
Abstract Cilj istraživanja: Francisella novicida je unutarstanična bakterija koja može inficirati brojne stanice uključujući i stanice ameba. Pretpostavlja se da amebe imaju važnu ulogu za održavanje francizela u vodenom okolišu. Za razliku od stanica sisavaca gdje citosolnom razmnožavanju ove bakterije prethodi kratki ulazak u fagosom, u stanicama ameba bakterija se razmnožava u vakuoli. Postavljena je hipoteza da boravak bakterije u fagosomu/vakuoli i u stanicama makrofaga i u stanicama ameba, ima bitnu ulogu u unutarstaničnom životu bakterije, njenoj virulenciji i patogenezi tularemije. Zbog toga su ciljevi rada bili: uspostaviti okolišnu amebu Dictyostelium discoideum kao modelni organizam za proučavanje unutarstaničnog života F. novicida, razviti metodu za izolaciju fagosoma/vakuole u kojima se nalazi F. novicida iz stanica D. discoideum i primarnih stanica humanih makrofaga, kao i identificirati proteine uključene u formiranje vakuole u kojoj se nalazi F. novicida nakon infekcije D. discoideum.
Materijal i metode: U ovom radu istražen je endosomalno – lizosomalni put F. novicida u D. discoideum metodama konfokalne i elektronske mikroskopije te Western blot analizom. U svrhu analize fagosoma/vakuole u kojima se nalazi F. novicida nakon infekcije stanica ameba i humanih makrofaga, uspostavljena je metoda za njihovu izolaciju. Identifikacija proteina koji su uključeni u sastav vakuole provedena je masenom spektrometrijom.
Rezultati: Praćenjem kinetike rasta pokazano je da se F. novicida razmnožava u D. discoideum. Bakterija se nakon ulaska u amebu nalazi u ranom vakuolarnom odjeljku, gdje se i razmnožava. Uspostavljena je metoda za izolaciju fagosoma/vakuole u kojoj se nalazi F. novicida i njihove membrane su visoko očuvane. Kvantitativnom proteomskom analizom uzoraka frakcije vakuola u kojima se nalazi F. novicida izoliranih iz D. discoideum 30 minuta nakon infekcije identificirano je ukupno 974 proteina s visokom grupnom specifičnošću.
Zaključak: Rezultati ovog istraživanja pokazali su da F. novicida raste i razmnožava se u D. discoideum te ameba može služiti kao modelni organizam za proučavanje virulencije vrsta Francisella i patogeneze tularemije. Bakterija se nakon infekcije razmnožava u vakuoli. Uspješno je uspostavljena metoda za izolaciju fagosoma/vakuole u kojoj se nalazi F. novicida nakon infekcije D. discoideum i humanih makrofaga. Masenom spektrometrijom identificirani su proteini rane vakuole endosomalno – lizosomalnog puta što dokazuje lokalizaciju bakterije u vakuoli.
Abstract (english) Objectives: Francisella novicida is an intracellular bacterium that can infect multiple range of cells, including amoeba cells. It has been suggested that amoeba have important role for survival of Francisella in the water environment. After infection of mammalian cells, bacterium enters the phagosome and escapes in the cytosol where it replicates. In contrast, in amoeba cells bacterium replicates in vacuoles. We hypothesised that localization of Francisella in the phagosome/vacuole in mammalian and amoeba cells has an important role in the intracellular life cycle of Francisella, bacterial virulence as well as the pathogenesis of tularemia. The aim of this study was to establish Dictyostelium discoideum as a model organism for studying intracellular life cycle of Francisella. Specific aims were to establish a method for isolation of phagosome/vacuole containing Francisella from D. discoideum and human macrophages, and identification of proteins important in formation of the vacuole containing Francisella after infection of D. discoideum.
Material and Methods: In this thesis, the endocytic pathway of F. novicida in D. discoideum was investigated using confocal microscopy, electron microscopy and Western blot analysis. The method for isolation of phagosome/vacuole containing Francisella was established. Proteins included in formation of vacuole containing Francisella were identified using mass spectrometry.
Results: Growth kinetics study showed that F. novicida replicates in D. discoideum. After entry, bacterium is localized in early vacuolar compartment where it replicates. The method for isolation of phagosome/vacuole containing Francisella enabled the isolation of highly intact vacuoles. Proteomic analysis of fraction with vacuoles containing Francisella isolated from D. discoideum, 30 minutes after infection, identified 974 proteins with high group confidence.
Conclusion: Results of this thesis showed that F. novicida replicates in D. discoideum, and amoeba can be used as a model organism for studying virulence of Francisella and pathogenesis of tularemia. After infection, bacterium replicates in the vacuoles. The method for isolation of phagosome/vacuole containing Francisella was successfully established. Mass spectrometry analysis identified proteins included in the formation of early vacuolar compartment in endocytic pathway, which confirmed localization of Francisella in vacuole.
Keywords
Dictyostelium
Fagosom
Francisella
Unutarstanični život.
Keywords (english)
Dictyostelium
Francisella
Intracellular lifestyle
Phagosome
Language croatian URN:NBN urn:nbn:hr:184:584082 Project Number: IP-2016-06-9003 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2020-02-21 09:30:32
