Blanc SVSE 8 - Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale

Iron uptake pathways in Pseudomonas aeruginosa: their specificities and complexity – IronPath

IronPath

This project belongs to the «Dynamics of Bacterial Membrane Proteins« network, which brings together 7 German teams, a Swiss team and my group. The research of all these groups focuses on the dynamics of membrane proteins in bacteria and use cell biology and biochemical/biophysical approaches. Within this international network, our group will investigate the membrane transport of iron by the sidérophores pyoverdine (Pvd) and pyochelin (Pch) in Pseudomonas aeruginosa.

Iron uptake pathways in pseudomonas aeruginosa: their specificitiés and complexity

We propose to focus on new aspects of ferrisiderophore transport, which disserved little or no attention until now, like the mechanism of iron release from the siderophore and iron transport across the inner membrane. FpvCDEF and FpvGHJK are two inner membrane complexes probably involved in this process. The originality of FpvGHJK is its involvement in iron release from Pvd and the absence of any homology with known reductases. FpvCDEF is an ABC transporter probably involved in the transport of iron across the inner membrane. Its particularity is the presence of two periplasmic binding proteins. The present project will focus on these new inner membrane protein complexes and we will try to understand by in vivo and in vitro approaches the molecular mechanism involved in their biological function. <br />Another major aim of this project will be to determine, in our model P. aeruginosa, the cellular localization and distribution of different enzymes involved in PVd and Pch biosynthesis and proteins involved in iron uptake. Moreover, the organization and especially the dynamics of the molecular interactions between the different proteins of the Pvd pathway will be investigated. Cell biology approaches, fluorescent-tagged proteins and fluorescent microscopy (FRET and FRAP) will be used. This study will provide information on how all these proteins interact in vivo and how they are organized and distributed within the cell membranes during bacterial growth. <br />With all these approaches, IronPath will provide an insight into the molecular interactions between the proteins involved in iron uptake in P. aeruginosa, their organization and distribution in the cell wall and how the resulting machin enables iron acquisition. Such a study has never been undertaken even for the well known iron uptake pathways of E. coli. <br />

The scientific program is divided into 7 sections (including management).
1. We will investigate using high resolution microscopy, the high degree of intracellular organization of Pvd and Pch biosynthesis and look for the existence of a “siderosome” and analyze its protein composition.
2. In parallel, with the same approach we will also investigate the cellular distribution and co-localization of the different proteins involved in iron uptake by Pvd and Pch in P. aeruginosa.
3. We will try to understand, using these fluorescent labeled mutants, which environmental factors make P. aeruginosa use rather Pvd than Pch or vise versa to get access to iron. The time necessary to switch from the use of one siderophore to another will be investigated as well.
4. The use of fluorescent fusion proteins will also make it possible to study the dynamics of protein movements in all cell compartments of living cells by FRAP (Fluorescence Recovery After Photobleaching).
5. The mechanism of iron release from Pvd in the periplasm will be investigated. This mechanism involves iron reduction and FpvCDEF acts probably as the iron reducer. The enzymatic mechanism involved in this process will be investigated in vivo and in vitro with a reconstituted system. The enzymatic reaction will be followed in vivo and in vitro using the fluorescence of Pvd (apo Pvd being fluorescent and Pvd-Fe being not; iron quenches the fluorescence of the chelator). The ability of the FpvCDEF system to dissociate metal other than iron from Pvd will be investigated as well. The role of FpvGHJK in Pvd-Fe dissociation will be investigated as well. Mutant (gene deletions, mutations of important residues) will be tested.
6. All the proteins involved in a siderophore pathway must form big machineries with different successive protein interactions. To understand the functioning of these complex systems, the Pvd-Fe uptake machinery will also be investigated as a whole in vivo and in vitro with purified proteins.

The first results are in progress and will be published here once the data have been published in a scientific journal.

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This project belongs to the "Dynamics of Bacterial Membrane Proteins" network, which brings together teams from the Universität of Freibourg, a team from the Ruprecht-Karls-Universität-Heidelberg, a Swiss team from the Biozentrum (Basel) and my group (Université de Strasbourg-CNRS). The research of all these groups focuses on the dynamics of membrane proteins in bacteria and use cell biology and biochemical/biophysical approaches. The German DFG (Deutsche Forschungsgemeinschaft) and the ANR funded this network the last three years. Last January (2011), this consortium received again a positive evaluation from the DFG for a new funding for 3 years. Because of administrative raisons, our group cannot be funded by the DFG and requires funding from a French organization.
Within this international network, our group will investigate the membrane transport of iron in Pseudomonas aeruginosa, an opportunistic human pathogen. Iron is essential for almost all living organisms but poorly soluble. To overcome this problem, microorganisms acquire this metal by secreting and then reabsorbing siderophores, low molecular mass compounds with a very high affinity for iron ions. P. aeruginosa produces two major siderophores pyoverdine (Pvd) and pyochelin (Pch). In Gram-negative bacteria, siderophore-mediated iron acquisition requires complex membrane protein machines that span the outer and the inner bacterial membranes. The last 20 years, iron uptake studies have been mostly focused on siderophore synthesis and ferrisiderophore uptake across the outer membranes, from the extracellular medium to the periplasm.
In IronPath, we propose to focus on new aspects of ferrisiderophore transport, which disserved little or no attention until now, like the mechanism of iron release from the siderophore and iron transport across the inner membrane. These studies will be carried out on P. aeruginosa because iron uptake in this bacteria is more complex than in E. coli and we have identified two new inner membrane protein complexes, FpvCDEF and FpvGHJK, involved in the Pvd pathway of P. aeruginosa and absent in E. coli. The originality of FpvGHJK is its involvement in iron release from Pvd and the absence of any homology with known reductases. FpvCDEF is an ABC transporter probably involved in the transport of iron across the inner membrane. Its particularity is the presence of two periplasmic binding proteins. The present project will focus on these new inner membrane protein complexes and we will try to understand the molecular mechanism involved in their biological function.
Another major aim of this project will be to determine, in P. aeruginosa, the cellular localization and distribution of different enzymes involved in Pvd and Pch biosynthesis and proteins involved in iron uptake (presence or absence of specific sites within and along bacterial membranes, periplasm and cytoplasm). Indeed, bacteria are now understood to have a higher degree of intracellular organization than previously thought, with individual proteins located at particular sites within the bacterial cell. This cellular organization has never been investigated for ferrisiderophore pathways in bacteria. The organization and especially the dynamics of the molecular interactions between the different proteins of the Pvd and Pch pathways will be investigated. Cell biology approaches, fluorescent-tagged proteins and fluorescent microscopy (FRET and FRAP) will be used. This study will provide information on how all these proteins interact in vivo and how they are organized and distributed within the cell membranes during bacterial growth.
With all these approaches, IronPath will provide an insight into the molecular interactions between the proteins involved in iron uptake in P. aeruginosa, their organization and distribution in the cell wall and how the resulting machine enables iron acquisition. Such a study has never been undertaken even for the well-known iron uptake pathways of E. coli.

Project coordinator

Madame Isabelle Schalk (Biotechnologie et Signalisation Cellulaire) – isabelle.schalk@unistra.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

UMR7242 Biotechnologie et Signalisation Cellulaire

Help of the ANR 299,000 euros
Beginning and duration of the scientific project: December 2012 - 36 Months

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