Title Uloga pilO gena u patogenezi eksperimentalne tularemije Title (english) The role of the pilO gene in experimental tularemia Author Mateja Ožanič Mentor Marina Šantić (mentor)Committee member Maja Abram (predsjednik povjerenstva)Committee member Ana Budimir (član povjerenstva)Committee member Vladimir Mićović (član povjerenstva)Granter University of Rijeka Defense date and country 2015, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences Abstract CILJEVI: Postavljena je hipoteza da gen koji je uključen u ekspresiju pila (pilO), ima bitnu ulogu u unutarstaničnom životu bakterije u makrofazima i amebama te u aktivaciji imunološkog odgovora i patogenezi tularemije u miševa. Ciljevi rada bili su odrediti važnost pilO gena u unutarstaničnom životu F. tularensis subsp. novicida u humanim makrofazima i amebama te u patogenezi tularemije u BALB/c miševa, kao i pratiti ulogu pilO gena u imunološkom odgovoru BALB/c miševa na infekciju bakterijom F. tularensis subsp. novicida.
METODE: Ovim je istražinjam praćeno unutarstanično razmnožavanje pilO mutante bakterije F. tularensis subsp. novicida u humanim makrofazima i Acanthamoeba castellanii. Konfokalnom mikroskopijom istraženo je kretanje pilO mutante uzduž endosomalno-lizosomalnog degradirajućeg puta u humanim makrofazima. Elektronskom mikroskopijom istražena je ultrastruktura humanih makrofaga i A. castellanii inficiranih s pilO mutantom. Praćena je lokalizacija pilO mutante unutar stanica kao i cjelovitost fagosomalne membrane. Ispitana je virulencija pilO mutante u in vivo modelu na BALB/c miševima nakon intratrahealne (i.t.) i indradermalne (i.d.) infekcije. Određena je kinetika rasta bakterija u organima miševa, a elektronskom mikroskopijom praćena je ultrastruktura organa. Svjetlosnom mikroskopijom istražene su patohistološke promjene u organima BALB/c miševa nakon intratrahealne i intradermalne infekcije pilO mutantom. Kako bi se ispitao imunološki odgovor miševa na infekciju pilO mutantom, analizirana je ekspresija citokina i kemokina u serumu inficiranih životinja.
REZULTATI: U usporedbi s divljim sojem bakterije F. tularensis subsp. novicida, pilO mutanta pokazala je značajno slabiju sposobnost razmnožavanja u humanim makrofazima i A. castellanii. Rezultati konfokalne i elektronske mikroskopije pokazali su kako za razliku od divljeg soja bakterije, pilO mutanta ne uspijeva pobijeći iz fagosoma u citosol humanih makrofaga. U stanicama A. castellanii, pilO mutanta se nalazi unutar membranama omeđenih vakuola, međutim ne dolazi do razmnožavanja bakterije. Bez obzira na put infekcije, u usporedbi s divljim sojem bakterije, pilO mutanta je atenuirana u modelu na miševima. Mutanta pilO nema sposobnost izlaska iz fagosoma i unutarstaničnog razmnožavanja u plućima, jetri i slezeni BALB/c miševa. Nadalje, pilO mutanta ne dovodi do većih patohistoloških promjena unutar organa inficiranih miševa. Prilikom infekcije BALB/c miševa pilO mutantom, u serumu miševa detektirane su statistički značajne razlike u ekspresiji citokina IFN-γ, TNF-α, IL-17, IL-12(p40), IL-12(p70), IL-1α, IL-1β, IL-10, G-CSF, GM-CSF u usporedbi s divljim sojem bakterije F. tularensis subsp. novicida. Nadalje, razine svih testiranih kemokina u serumu miševa inficiranih pilO mutantom bile su niže u usporedbi sa serumom životinja inficiranih divljim sojem bakterije.
ZAKLJUČCI: Rezultati ovog istraživanja pokazali su kako pilO gen F. tularensis subsp. novicida ima važnu ulogu u bijegu bakterije iz fagosoma u citosol humanih makrofaga te u unutarstaničnom razmnožavanju. Gen pilO neophodan je i za razmnožavanje F. tularensis subsp. novicida unutar A. castellanii. Nadalje, pilO gen važan je za virulenciju F. tularensis subsp. novicida u BALB/c miševa. Taj gen ima važnu ulogu u patogenezi tularemije u plućima, jetri i slezeni miševa, kao i u imunološkom odgovoru životinja na intratrahealnu i intradermalnu infekciju bakterijom F. tularensis subsp. novicida
Abstract (english) OBJECTIVES. The aim of this study was to determine the role of the pilO gene in intracellular life of F. tularensis subsp. novicida within human macrophages and Acanthamoeba castellanii. Further, we wanted to determine the role the pilO gene in the pathogenesis of tularemia in BALB/c mice as well as in immune responce of BALB/c mice.
METHODS: In the present study, growth kinetics of the pilO mutant was determined in human monocyte derived macrophages and A. castellani. Confocoal microscopy analysis was used to study the trafficking of the pilO mutant along the endocytic pathway within the human monocyte derived macrophages. Transmission electron microscopy was performed to study the intracellular localization of the pilO mutant within human monocyte derived macrophages and A. castellani as well as the intergrity of the phagosomal membrane. Further, we examined survival of BALB/c mice and bacterial burdens in lungs, liver and spleen following intratracheal and intradermal infection with the pilO mutant. Histopathology of lungs, liver and spleen of infected BALB/c was analysed using light microscopy. The ultrastructure of lungs and spleen of infected BALB/c mice was examined by transmission electron microscopy. In addition, we examined the immune response in BALB/c mice infected by i.t. and i.d. route with the pilO mutant strain. Chemokine and cytokine expression was analysed in serums of infected BALB/c mice.
RESULTS: In comparison to the wild type F. tularensis subsp. novicida, pilO mutant showed significantly lower intracellular replication within human monocyte derived macrophages and A. castellanii. Results from confocoal and transmission electron microscopy analysis showed that pilO mutant does not escape the phagosom and therefore is defective for replication in human macrophages. Mutant pilO was not able to replicate within A. castellanii and single bacteria were localized within membrane bound vacuoles. Further, pilO mutant strain was attenuated for virulence in BALB/c mice. This mutant was defective for intracellular replication in the lungs, liver and spleen of BALB/c mice, and no histopathological changes were observed within organs of infected animals. Serum levels of cytokines IFN-γ, TNF-α, IL-17, IL-12(p40), IL-12(p70), IL-1α, IL-1β, IL-10, G-CSF and GM-CSF were signifficantly different following infection of mice with the pilO mutant strain, in comparison to the wt strain. Further, serum levels of all tested chemokines in serum of BALB/c mice were lower after infection with pilO mutant strain in comparison to the wild type F. tularensis subsp. novicida.
CONCLUSIONS: This study showed that pilO gene plays an important role in phagosomal escape and intracellular replication of F. tularensis subsp. novicida in human monocyte derived macrophages. Gene pilO is also essential for replication of this bacterium within A. castellanii. Further, pilO gen is important for virulence of F. tularensis subsp. novicida in BALB/c mice. Gene pilO plays an important role in pathogenesis of tularemia witnin lungs, liver and spleen of BALB/c mice, as well as in immune responce of BALB/c mice to intratracheal and intradermal infection with F. tularensis subsp. novicida.
Keywords
Amebe
Francisella tularensis subsp. novicida
Humani makrofazi
Miševi
pilO mutanta
Keywords (english)
Amoebae
Francisella tularensis subsp. novicida
Human macrophages
Mice
pilO mutant
Language croatian URN:NBN urn:nbn:hr:184:019073 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 2023-01-18 12:37:54
