Molecular mechanism involved in the iron acquisition via pyoverdine and desferrichrome in Pseudomonas Aeruginosa
Molecular mechanism involved in the iron acquisition via pyoverdine and desferrichrome in Pseudomonas Aeruginosa.
Thèses de doctorat,
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Iron is essential for growth of mostly all microorganisms. Despite it’s high abundance on earth, iron present a low bioavailability at physiological pH. To over come this low iron availability, many microorganisms produce and secrete siderophore. Siderophore are low molecular weight molecules having a high affinity for iron. Our bacterium model, Pseudomonas aeruginosa produces two siderophores : pyoverdine (Pvd) and pyochelin. These two iron uptake mechanisms implies many proteins involved in the biosynthesis of siderophore and the secretion in the extracellular medium of the siderophores. They are also involved in the mechanism of iron acquisition via siderophores and in the regulation of the system. During my PhD, we have studied the involvement of the PvdRT-OpmQ efflux pump in the secretion of de novo synthesized Pvd. Previous studies had shown that this ATP type efflux pump was involved in the recycling of Pvd into the extracellular medium after iron release in the periplasmic space. By using molecular and cellular techniques (fluorescent microscopy and cellular fractionnation) and the fluorescent properties of Pvd, we have shown that the PvdRT-OpmQ efflux pump was also involved in the secretion of de novo synthesized Pvd. With the same approach, we were also able to show that this same efflux pump was responsible for the control of the selectivity of the Pvd pathway torward iron. Indeed, Pvd is able to bound different metals in the extracellular environment. We have shown using ICP-AES, that the different Pvd-metal complexes are transported through the outer membrane receptor FpvA but all the complexes except Pvd-Fe complex are immediately expelled into the extracellular medium by PvdRT-OpmQ. In absence of PvdRT-OpmQ, when P. aeruginosa is incubated with different Pvd-metal complexes, they accumulate in the periplasm of the bacterium. We also tried to understand the mechanisms which are involved in the transport of the Pvd-Fe complex through the outer membrane receptor FpvA. FpvA is composed of a 22 beta barrel strand closed by a plug. We tried to understand the mechanism of chanel formation allowing the transport of Pvd-Fe. For this study, we used the properties of Pvd to do FRET with the Tryptophanes (Trp) of the proteins. FpvA has 17 Trps in his sequence but they are all localized in the beta barrel. In this study, we inserted Trp in the plug of the receptor by site directed mutagenesis in the purpose to follow the interaction between this domain and Pvd-Fe. Finally, we have studied the iron acquisition via desferrichrome in P. aeruginosa. When I began my PhD, only the outer membrane receptor had been identified by proteomic studies. We used single mutants from the washington collection to do this study. We have clearly identified the outer and inner membrane transporter and also the enzyme involved in the dissociation of the ferrichrome in the cytoplasm. A fluorescent analogue of ferrichrome was also used to do all the dissociation kinetics usually used in the laboratory.
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