Title ULOGA ATG5 U PATOGENEZI EKSPERIMENTALNE
TULAREMIJE Title (english) THE ROLE OF ATG5 IN THE PATHOGENESIS OF
EXPERIMENTAL TULAREMIA Author Ina Viduka Mentor Marina Šantić (mentor)Committee member Irena Slavuljica (predsjednik povjerenstva)Committee member Ana Budimir (član povjerenstva)Committee member Darinka Vučković (član povjerenstva)Granter University of Rijeka Defense date and country 2022, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences Abstract Cilj istraživanja: Unutarstanični patogen Francisella tularensis vrlo brzo nakon
infekcije pobjegne iz fagosoma u citosol gdje se nesmetano razmnožava
izbjegavajući razgradnju putem autofagije. Prijašnje su studije pokazale da
Francisella izbjegava autofagiju, ali nije u potpunosti razjašnjeno koristi li ju za svoje
unutarstanično razmnožavanje. Osim toga, uloga autofagije u patogenezi tularemije
još uvijek nije istražena in vivo. Cilj ovog istraživanja bio je ispitati ulogu ATG5-ovisne
autofagije u unutarstaničnom životu F. tularensis subsp. holarctica soj LVS koristeći
in vitro i in vivo modele infekcije.
Materijal i metode: Ulogu ATG5-ovisne autofagije in vitro ispitali smo praćenjem
unutarstaničnog razmnožavanja i citotoksičnog učinka F. tularensis subsp. holarctica
soj LVS u primarnim humanim i primarnim mišjim makrofagima te primarnim ATG5-
deficijentnim mišjim makrofagima. Konfokalnom i elektronskom mikroskopijom ispitali
smo interakciju bakterija s autofagičnim vakuolama. U svrhu in vivo istraživanja,
uspostavili smo miševe s nedostatkom ATG5 u mijeloidnoj liniji. Nakon intradermalne
infekcije ATG5-deficijentnih miševa sa sojem LVS, pratili smo preživljavanje miševa,
ispitali broj bakterija i patohistološke promjene u organima inficiranih miševa te
analizirali lokalizaciju bakterija u tkivima metodom elektronske mikroskopije.
Rezultati: Farmakološki inhibitori autofagije i nedostatak ATG5 statistički su smanjili
unutarstanično razmnožavanje i citotoksični učinak F. tularensis subsp. holarctica
soja LVS u primarnim mišjim i primarnim humanim makrofagima. Rezultati
konfokalne i elektronske mikroskopije potvrdili su da je ATG5 odgovoran za
usmjeravanje Francisella u autofagosome 24 sata nakon infekcije ubikvitin-SQSTM1-
LC3 putem. Infekcija ATG5-deficijentnih miševa sojem LVS rezultirala je povećanim
preživljavanjem, značajno smanjenim razmnožavanjem bakterija u organima miševa
te blažim patohistološkim promjenama u tkivu jetre, slezene i pluća.
Zaključak: F. tularensis subsp. holarctica soj LVS inducira probakterijsku autofagiju
te iskorištava dobivene nutrijente za svoje unutarstanično razmnožavanje u in vitro i
in vivo modelu infekcije. ATG5 ima važnu ulogu u eksperimentalnoj tularemiji budući
da usmjerava LVS soj u autofagične vakuole što podržava njeno unutarstanično
razmnožavanje.
Ključne riječi: Autofagija; Francisella; Makrofagi; Miševi; Tularemija
Abstract (english) Objectives: After infection, the intracellular pathogen F. tularensis subsp. holarctica
strain LVS rapidly escapes from the phagosome into the cytosol, where the
bacterium proliferates without degradation by autophagy. Previous studies have
shown that Francisella avoids autophagy degradation, but it is not yet fully
understood whether the bacteria obtain nutrients from autophagy that support their
intracellular replication. The role of autophagy in the pathogenesis of tularemia in vivo
is also unknown. The main objective of this study is to investigate the role of ATG5-
dependent autophagy in the intracellular life of F. tularensis subsp. holarctica strain
LVS using in vitro and in vivo models.
Material and methods: To investigate the role of autophagy in vitro, we examined
intracellular replication and cytotoxicity of F. tularensis subsp. holarctica strain LVS in
primary murine and primary human macrophages, and in ATG5-deficient murine
macrophages. We investigated the interaction between Francisella and autophagic
vacuoles using confocal and electron microscopy. For in vivo experiments, we bred
mice with ATG5 deficiencies in the myeloid lineage. We infected ATG5-deficient mice
intradermally with the strain LVS and examined mouse survival, bacterial burden, and
histopathological changes in the organs of the infected mice. We also analyzed the
localization of bacteria in the tissues of the mice using electron microscopy.
Results: Pharmacological auophagy inhibitors and deficiency of ATG5 resulted in
decreased intracellular replication and cytotoxicity of F. tularensis subsp. holarctica
strain LVS in primary murine macrophages and primary human macrophages. Our
results from confocal and electron microscopy show that ATG5 is responsible for
targeting Francisella in autophagosomes 24 hours after infection via the ubiquitinSQSTM1-LC3-dependent pathway. Infection of ATG5-deficient mice with the strain
LVS resulted in increased survival, decreased bacterial burden, and less severe
histopathological changes in liver, spleen, and lung tissues.
Conclusion: F. tularensis subsp. holarctica strain LVS induces probacterial
autophagy and uses the extracted nutrients to ensure its proliferation in vivo and in
vitro. ATG5 plays an important role in experimental tularemia by directing the LVS
strain into autophagic vacuoles that support its intracellular proliferation.
Keywords
Autofagija
Francisella
Makrofagi
Miševi
Tularemija
Keywords (english)
Autophagy
Francisella
Macrophages
Mice
Tularemia
Language croatian URN:NBN urn:nbn:hr:184:836442 Project Number: IP-2016-06-9003 Project Number: uniri-biomed-18-128 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 10:55:02
