Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins

• Published in: International journal of molecular sciences Vol. 21; no. 15; p. 5496
• Main Authors: Siebert, Claire, Mercier, Corinne, Martin, Donald K, Renesto, Patricia, Schaack, Beatrice
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 31.07.2020; MDPI
• Subjects: Amino acids; Antibiotics; Bacterial Proteins - genetics; Bacterial Vaccines; Bacteriology; Biochemistry, Molecular Biology; Biological Transport - genetics; Biological Transport - immunology; Biological Warfare Agents; Conductance; Drug resistance; Electrophysiology; Escherichia coli - genetics; Fluorescence; fluorescence flux; Fluoroquinolones; Fluoroquinolones - adverse effects; Fluoroquinolones - therapeutic use; Francisella tularensis; Francisella tularensis - genetics; Francisella tularensis - metabolism; Francisella tularensis - pathogenicity; FupA; FupB; Fusion protein; Humans; impedance spectroscopy; Infections; Iron; Iron - metabolism; Life Sciences; Lipid bilayers; Lipid membranes; Lipids; Membrane vesicles; Membranes; Metabolism; Microbiology and Parasitology; Molecular modelling; Pore formation; Porins; Porins - genetics; Porins - metabolism; Protein transport; Proteins; Resistance; Structural Biology; Sucrose; Tularemia; Tularemia - drug therapy; Tularemia - genetics; Tularemia - microbiology; Vaccines; Virology; Virulence; FupB; FupA; fluorescence flux; impedance spectroscopy; porins; Francisella tularensis
• DOI: 10.3390/ijms21155496
• ISSN: 1422-0067; 1661-6596

Responsible for tularemia, 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 deletion contributed to lower 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 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 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 infections.