Abstract
Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.
Highlights
The facultative intracellular Gram-negative coccobacillus Francisella tularensis is the etiologic agent of tularemia [1]
The name FupA (Fer Utilization Protein A) was proposed for this 58 kDa protein since it was very similar to the F. tularensis siderophore receptor FslE that was required for the acquisition of siderophore-bound iron [6]
From the observation that FupA expression is regulated neither by the ferric uptake regulator (Fur) protein nor by the iron level, it was suggested that this protein may not be involved in iron acquisition and that it could instead be involved in the transport of additional substrates [10]
Summary
The facultative intracellular Gram-negative coccobacillus Francisella tularensis is the etiologic agent of tularemia [1]. From the observation that FupA expression is regulated neither by the ferric uptake regulator (Fur) protein nor by the iron level, it was suggested that this protein may not be involved in iron acquisition and that it could instead be involved in the transport of additional substrates [10] Among such unexpected functions, we demonstrated that the deletion of fupA or fupA/B contributes to reduced antibiotic susceptibility and could promote the emergence of antibiotic resistance mediated by an increased biofilm formation [9]. FupA and FupA/B have been reported to facilitate iron transport [10,13,14] From these data, we hypothesized that these three Francisella OMPs, which are partially folded as β-barrels, assemble to form channel-forming transmembrane porins. Using fluorescent flux assays and electrophysiology, we demonstrated that FupA, FupB, as well as FupA/B assemble as functionally active porins when correctly folded in a lipid environment
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
