Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins

International audienceResponsible 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

Mechanism of action of the moonlighting protein EfTu as a Substance P sensor in Bacillus cereus

Abstract The striking feature of the ubiquitous protein EfTu (Thermo unstable ribosomal Elongation factor) is its moonlighting (multifunctional) activity. Beyond its function at the ribosomal level it should be exported to the bacterial surface and act as an environmental sensor. In Bacillus cereus, and other cutaneous bacteria, it serves as a Substance P (SP) receptor and is essential for bacterial adaptation to the host. However, the modus operandi of EfTu as a bacterial sensor remains to be investigated. Studies realized by confocal and transmission electron microscopy revealed that, in the absence of an exogenous signal, EfTu is not exposed on the bacterial surface but is recruited under the effect of SP. In addition, SP acts as a transcriptional regulator of the tuf gene encoding for EfTu. As observed using gadolinium chloride, an inhibitor of membrane mechanosensitive channels (Msc), Msc control EfTu export and subsequently the bacterial response to SP both in terms of cytotoxicity and biofilm formation activity. Microscale thermophoresis revealed that in response to SP, EfTu can form homopolymers. This event should occur after EfTu export and, as shown by proteo-liposome reconstruction studies, SP appears to promote EfTu polymers association to the membrane, leading subsequently to the bacterial response. Molecular modeling suggests that this mechanism should involve EfTu unfolding and insertion into the bacterial cytoplasmic membrane, presumably through formation of homopolymers. This study is unraveling the original mechanism action of EfTu as a bacterial sensor but also reveals that this protein should have a broader role, including in eukaryotes

Metabolism: a Bottleneck in in Vitro Toxicological Test Development. The Report and Recommendations of ECVAM Workshop 54

Abstract is not availableJRC.I.2-Validation of biomedical testing method