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Deletion of the Major Facilitator Superfamily Transporter fptB Alters Host Cell Interactions and Attenuates 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. The fact 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, has 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 the major facilitator superfamily (MFS) transporter fptB Based on the attenuating capacity of this deletion in the F. tularensis LVS background, we hypothesized that the deletion of this transporter would alter the intracellular replication and cytokine induction of the type A strain and attenuate virulence in the stringent C57BL/6J mouse model. Here we demonstrate that the deletion of fptB significantly alters the intracellular life cycle of F. tularensis, attenuating intracellular replication in both cell line-derived and primary macrophages and inducing a novel cytosolic escape delay. Additionally, we observed prominent differences in the in vitro cytokine profiles 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.

SUBMITTER: Balzano PM

PROVIDER: S-EPMC5820938 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb3-Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. IMPORTANCE Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.

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Deletion of the Major Facilitator Superfamily Transporter fptB Alters Host Cell Interactions and Attenuates Virulence of Type A Francisella tularensis.

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