The role of iglI mutant of Francisella tularensis LVS strain in experimental tularemia

Francisella tularensis je gram-negativna unutarstanična bakterija. Pripada porodici Francisellaceae i uključuje 3 podvrste: tularensis, holarctica i mediasiatica koje se međusobno razlikuju u virulenciji, geografskoj rasprostranjenosti i ekologiji. Francisella je uzročnik tularemije, re-emergentne bolesti ljudi, domaćih i divljih životinja. Francisella ima sposobnost inficirati makrofage, nakon čega različitim mehanizmima koji trenutno nisu u potpunosti definirani, bježi iz fagosoma te se opsežno replicira u citosolu. Živi atenuirani soj LVS (engl. Live Vaccine Strain), koji je dobiven iz podvrste holarctica, nije virulentan za ljude no vrlo je virulentan za miševe stoga je idealan za proučavanje mehanizma virulencije roda Francisella. U stanicama sisavaca veliku ulogu za preživljavanje i replikaciju te posljedično virulenciju Francisella imaju geni koji se nalaze unutar Francisella patogenog otoka (engl. Francisella pathogenicity island, FPI). Gen iglI neophodan je za virulenciju, citotoksičnost i bijeg iz fagosoma. Cilj ovog diplomskog rada bio je utvrditi ulogu iglI gena in vivo praćenjem preživljavanja i određivanjem broja bakterija u organima intradermalno inficiranih miševa. Također, ispitivao se i imunološki odgovor domaćina sintezom interferon gamma (IFN-γ) i interleukin 10 (IL-10) citokina, in vivo na C57BL/6 miševima nakon intradermalne infekcije s F. tularensis subsp. holarctica LVS, mutantom iglI i njenom komplementantom iglI/iglI. Kod testa preživljavanja miševa, tijekom 15 dana praćeno je 10 miševa po skupini koji su intradermalno inficirani. Za određivanje broja bakterija u tkivu jetre, slezene i pluća, 3 miša po skupini inficirani su s LD50 (5 x 104 bakterija/mišu), te su 6, 24, 48, 72 sata nakon infekcije organi odstranjeni i dalje analizirani. qRT-PCR-om određena je razina IFN-γ i IL-10 citokina u tkivu jetre, slezene i pluća 72 sata nakon intradermalne infekcije miševa kako bi se ispitao odnos između mutante iglI, divljeg soja LVS te komplementante iglI/iglI. Miševi inficirani iglI mutantom imali su 100% preživljavanje kod svih ispitivanih doza što se ne može reći za miševe inficirane s F. tularensis subsp. holarctica LVS. Također, broj bakterija mutante iglI u svim ispitivanim organima bio je statistički značajno smanjen u odnosu na soj LVS. Zaključno možemo reći kako je iglI gen važan za unutarstaničnu replikaciju F. tularensis subsp. holarctica LVS i da ima utjecaj na sintezu IFN-γ i IL-10 citokina u tkivu jetre, slezene i pluća.Francisella tularensis is a gram-negative intracellular bacteria. It belongs to the family Francisellaceae and includes three subspecies: tularensis, holarctica and mediasiatica which differ in virulence, geographical distribution and ecology. Francisella is the causative agent of tularemia, a re-emerging disease of humans, domestic, and wild animals. Francisella has the ability to infect macrophages, whereupon escapes from phagosomes by various mechanisms that are not currently fully defined, and replicates extensively in the cytosol. The live attenuated strain LVS (Live Vaccine Strain), produced from the subspecies holarctica, is not virulent to humans but is very virulent to mice and is therefore ideal for studying the virulence mechanism of the genus Francisella. In mammalian cells, Francisella pathogenicity island (FPI) genes play a major role in the survival, replication, and consequent virulence of Francisella. The iglI gene is essential for virulence, cytotoxicity, and phagosome escape. The aim of this thesis was to determine the role of iglI gene in vivo by monitoring survival and determining the number of bacteria in organs of intradermally infected mice. The immune response of the host was also examined by synthesis of interferon gamma (IFN-γ) and interleukin 10 (IL-10) cytokines, in vivo in C57BL/6 mice after intradermal infection with F. tularensis subsp. holarctica LVS, iglI mutant and its complement iglI/iglI. In the mice survival test, 10 mice per group of intradermally infected were followed for 15 days. To determine the number of bacteria in the liver, spleen and lungs tissue, 3 mice per group were infected with LD50 (5 x 104 bacteria/mouse), and the organs were removed 6, 24, 48, 72 hours after infection and further analyzed. The levels of IFN-γ and IL-10 cytokines in liver, spleen and lungs tissue were determined by qRT-PCR, 72 hours after intradermal infection of mice to examine the relationship between iglI mutant, wild-type LVS and complement iglI/iglI. Mice infected with the iglI mutant had 100% survival at all tested doses, which cannot be said for mice infected with F. tularensis subsp. holarctica LVS. Also, the number of iglI mutant bacteria in all examined organs was statistically significantly reduced compared to the LVS strain. In conclusion, the iglI gene is important for intracellular replication of F. tularensis subsp. holarctica LVS and that it affects the synthesis of IFN-γ and IL-10 cytokines in liver, spleen and lungs tissues