<i>Francisella tularensis</i>: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation

<i>Francisella tularensis</i> is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using <i>F. tularensis</i> live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain <i>F. tularensis</i> subsp. <i>tularensis</i> SCHU S4 deleted for the homologous FupA protein exhibited even higher FQ resistance. In addition to an increased FQ minimal inhibitory concentration, LVSΔ<i>fupA/B</i> displayed tolerance toward bactericidal compounds including ciprofloxacin and gentamicin. Interestingly, the FupA/B deletion was found to promote increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of vesicles from LVS and LVS∆<i>fupA/B</i> identified 801 proteins, including a subset of 23 proteins exhibiting differential abundance between both strains which may therefore contribute to the reduced antibiotic susceptibility of the FupA/B-deleted strain. We also demonstrated that OMVs are key structural elements of LVSΔ<i>fupA/B</i> biofilms providing protection against FQ. These results provide a new basis for understanding and tackling antibiotic resistance and/or persistence of <i>Francisella</i> and other pathogenic members of the <i>Thiotrichales</i> class.