Deletion of the MFS transporter fptB alters host cell interactions and attenuates the virulence of Type A Francisella tularensis

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 the handling of infected animals. That a very 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 Category A select agent and has renewed interest in the formulation of a vaccine. To this end, we engineered a Type A strain SchuS4 derivative containing a targeted deletion of major facilitator superfamily (MFS) transporter fptB. Based on the attenuating capacity of this deletion in the F. tularensis LVS background, we hypothesized that deletion of this transporter would alter intracellular replication and cytokine induction of the Type A strain and attenuate virulence in the stringent C57BL/6J mouse model. Here we demonstrate that deletion of fptB significantly alters the intracellular lifecycle of F. tularensis, attenuating intracellular replication in both cell line and primary macrophages, and inducing a novel cytosolic escape delay. Additionally, we observed prominent differences in the in vitro cytokine profile in human macrophage-like cells. The mutant was highly attenuated in the C57BL/6J mouse model, and provided partial protection against virulent Type A F. tularensis challenge. These results indicate a fundamental necessity for this nutrient transporter in the timely progression of F. tularensis through its replication cycle, and in pathogenesis.