Claire Siebert PhD Defense on 10/25/2018 - 01:30pm

PhD Defense of Claire SIEBERT from TheREx team on october the 25th at 01:30 pm :

« Molecular characterization of Francisella tularensis resistance to fluoroquinolones »


Place : Salle des Thèses, Bâtiment Boucherle, Faculté de Pharmacie Grenoble


Jury and thesis supervisor:

  •     Dr Patricia RENESTO, DR-CNRS, TIMC-IMAG, Director
  •     Dr Sandrine BOISSET, MCU-PH, Université Grenoble Alpes, Codirector
     
  •     Dr Thomas HENRY, DR-INSERM, CIRI, Reporter
  •     Dr Jean-Michel JAULT, DR-CNRS, IBCP, Reporter
  •     Dr Sophie JARRAUD, MCU-PH, Université Lyon I, Examiner
  •     Pr Patrice FAURE, PU-PH, Université Grenoble Alpes, Examiner
     

Abstract :

Francisella tularensis is a highly pathogenic Gram-negative bacterium responsible for Tularemia, a disease for which fluoroquinolones (FQ) are prescribed as the first-line therapy. The high rate of treatment failures and relapses observed despite appropriate treatment is a main concern. The emergence of FQ resistant bacteria has been thus hypothesized and a better characterization of the molecular mechanisms of bacterial resistance would be helpful.

In a first part, I analyzed the functional consequences of mutations on the DNA gyrase (FQ target) observed on F. novicida isolates highly-resistant to FQ and generated in vitro. The data obtained, based on supercoiling and DNA cleavage assays performed using recombinant gyrase subunits, clearly demonstrated that mutations on GyrA and GyrB were not accounting alone for FQ resistance.

In a second part, and based on the genomic analysis of FQ resistant mutants of F. tularensis subsp holarctica LVS, I considered another potential actor of FQ resistance, namely the FupA/B protein. Several approaches were thus carried out to characterize the role of this protein found to be inactivated in FQ resistant isolates. Using trans-complementation/mutagenesis approaches, we demonstrated that the expression of FupA/B is indeed related to the FQ sensitivity, thus validating our hypothesis. In addition, we have shown that the virulent strain F. tularensis subsp tularensis SCHU S4, lacking the FupA homologous protein, also had a lower FQ sensitivity. The deletion of FupA/B lipoprotein promotes increased secretion of "Outer Membrane Vesicles" (OMVs). The mass spectrometry proteomic analysis of the OMVs from the LVS and LVS-ΔfupA/B strains allowed the identification of 801 proteins among which a subset of 23 proteins of differential abundance between both strains. The observed changes could contribute to the increase in FQ sensitivity. Importantly, we observed that the overexpression of RecA was coupled with a higher tolerance of LVS-ΔfupA/B not only to ciprofloxacin, but also to gentamicin. Finally, we have shown that OMVs are structural key elements of F. tularensis biofilms known to provide protection against FQ.

Together, these results indicate that mutations targeting FupA/B contribute to antibiotic resistance through quantitative and qualitative modulations of OMVs production and biofilm formation. All these results open the way for exploring new pathways that would be helpful to combat Francisella resistance and / or persistence against antibiotics.

 

Key-words

Francisella, fluoroquinolones, resistance, DNA gyrase, OMVs, Biofilm