Abstract EN:

Pseudomonas aeruginosa is Gram negative opportunistic human pathogen that infects injured, immunodeficient and otherwise compromised patients. Under conditions of iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore named pyoverdine (Pvd) that chelates iron in the extracellular medium. The Pvd-Fe complex is then transported into the bacterium via the outer membrane TonB dependent receptor termed FpvA. This receptor consists of a membrane-spanning b-barrel occluded by a plug domain, which in its resting state leaves no space for an iron-loaded siderophore to pass through. The N-terminal extension of FpvA is involved in a signaling pathway that regulates the expression of the FpvA protein and its siderophore. Although the crystal structure of FpvA receptor is solved, the transfer mechanism of Pvd-Fe complex across the outer membrane of the bacteria has not been determined so far. A two-gated system has been proposed for the translocation of ferric-siderophore across the outer membrane. The first gate consists of the extracellular loops trapping Pvd-Fe in its binding site, and the second consists of the plug closing the barrel. The opening of the second gate is an energy-consuming process involving the inner membrane TonB complex. During my Ph. D thesis I studied the transport mechanism through the TonB dependent outer membrane receptor FpvA, and I characterized a second receptor termed FpvB The molecular determinants for specificity in Pvd-FpvA interactions were investigated through structural and biochemical approaches. The extracellular loops involved in the recognition of the Pvd-iron complex were identified using site directed mutagenesis, time-resolved fluorescence spectroscopy, and Fluorescence Resonance Energy Transfer (FRET). To get more insight on the translocation mechanism through the FpvA receptor, the ability of a reconstituted FpvA receptor obtained by cosynthesis of separately encoded plug and barrel domains to bind and transports Pvd-Fe was studied. Furthermore, the FpvA signaling, plug and barrel domains were characterized in vivo and the in vitro interactions between the purified FpvA domains and the proteins involved in the iron uptake machinery notably the periplasmic part of the TonB protein were studied. An additional pyoverdine receptor termed FpvB was described recently. Little is known about the Pvd-Fe uptake mechanism via this receptor, therefore FpvB was characterized at the molecular and functional level. FpvA and FpvB receptors are differentially expressed, and their expression is regulated differently. We showed that FpvB receptor transports Pvd-Fe with the same affinity as FpvA but, the identification of the elements involved in the uptake across the inner membrane remains to be identified combining molecular and functional approaches.