The Role of MFS Family Transporters in the Lifecycle of Type A Francisella tularensis and Their Viability as Vaccine Targets

Abstract

Francisella tularensis is a Gram negative facultative intracellular coccobacillus that can infect a wide variety of hosts. In humans, F. tularensis causes the zoonosis tularemia following insect bites, ingestion, inhalation, and handling infected animals. That a small inoculum delivered by the aerosol route can cause severe disease, coupled with the possibility of its use as an aerosolized bioweapon, have led to the classification of Francisella tularensis as a Tier 1 Category A select agent and has renewed interest in vaccine formulation. To this end, this study aimed to engineer and evaluate novel F. tularensis strain SchuS4 derivatives containing single deletions of major facilitator superfamily (MFS) transporters fptG, fptE, fptF, and fptB. Additionally, these strains developed for vaccine purposes were utilized to study more intimately aspects of the intracellular lifecycle of F. tularensis. Alterations to the intracellular lifecycle of the bacterium were measured using intracellular survival assays in multiple cell types. Two strains, F. tularensis SchuS4ΔfptB and SchuS4ΔfptG, displayed lifecycle alterations. Further analysis with these two strains involved characterization of the early inflammatory immune response from macrophages using ELISAs for cytokine secretion, and measuring alterations to host cell permeability by measuring supernatant lactate dehydrogenase release. In addition to in vitro analysis, F. tularensis strains SchuS4ΔfptB and SchuS4ΔfptG were evaluated for attenuation, and later, protective capacity, in vivo in the C57BL/6J mouse model. In vitro studies demonstrated that F. tularensis strains SchuS4ΔfptB and SchuS4ΔfptG were significantly altered in their intracellular lifecycle, exhibiting a novel cytosolic escape delay phenotype that was found to be autophagy-dependent for SchuS4ΔfptG. Additionally, F. tularensis strain SchuS4ΔfptB exhibited a significant replication defect. Moreover, we observed prominent differences in in vitro cytokine profiles in human and mouse macrophages for both mutants. Both mutants were attenuated in the C57BL/6J mouse model, and one strain, SchuS4ΔfptB, provided partial protection against virulent Type A F. tularensis challenge. These results demonstrate a fundamental necessity for these nutrient transporters in the timely progression of F. tularensis through its replication cycle, and in pathogenesis. Collectively, these strains validate the strategy of targeting transporters as means of attenuation for F. tularensis vaccine strain construction.