Ionen- und Antibiotika-Transport durch die Phosphat-spezifischen Porine OprP und OprO der äußeren Membran
Zusammenfassung der Projektergebnisse
Gram-negative bacteria contain the outer membrane as outermost barrier which, for survival, needs to allow the exchange of substrates with its environment. This outer membrane acts as a molecular sieve for water-soluble molecules. On the one hand, ubiquitous bacteria like Escheria coli contain wide pores allowing the passive diffusion of solutes based on their size and charge. On the other hand, certain bacteria include only narrow substrate-specific pores and among them is the bacterium Pseudomonas aeruginosa. Two of the pores from the latter bacterium are OprP and OprO which are induced under phosphate-starvation conditions and which were at the focus of the present project. While in a prior project the so-called arginine ladder within OprP was the main topic, this project partially focused on the lysine cluster located in the constriction zone and towards the periplasmic side for both membrane channels OprP and OprO. The effect of alanine mutations of individual lysine residues was investigated using electrophysiology and free energy calculations. All obtained data demonstrated that the closer the mutated lysine residues are to a specific arginine residue close to the center of the channel, the lower gets the single channel conductance. Concerning the permeation of antibiotics, some preliminary computational investigations had to be performed before going to the more ambitious free energy calculations concerning the antibiotics translocation through OprP and OrpO. To this end, a study was performed for the transport of fosfomycin permeation through the Outer Membrane Porin OmpF from E. coli. This is a rather wide pore and thus easier to simulate while the antibiotic molecule contains a phosphonic acid group and thus a likely candidate for the transport through OprP and OprO. The transport of fosfomycin through OmpF was determined using applied-field simulations, free energy calculations and measured using electrophysiology. A qualitative and to some extent quantitative agreement between all these techniques was achieved. The main body of the work was on the transport of fosfomycin and fosmidomycin, i.e., antibiotics molecules containing a phosphonic acid group, through the phosphate-specific pores OprP and OprO. From the computational side this was a formidable task since simulating the transport of molecules through narrow nanopores is not yet standard. That is why in a first study, we looked at the numerical convergence of different methods to obtain the energy landscape for the molecules permeating through OprP and OprO. To this end, the complexity of the permeating entities was increased from a simple chloride ions over inorganic phosphate to the antibiotic fosmidomycin. Armed with the experience from this investigation, the permeation of monophosphate, fosfomycin, and fosmidomycin through OprP and OprO was determined and analyzed. On the experimental side, we were able to demonstrate that fosfomycin and fosmidomycin are able to bind to the phosphate-specific binding sites inside the two pores. Moreover, the inhibition of chloride conductance of OprP and OprO by fosfomycin and fosmidomycin was measured, and it was concluded that both phosphonic acid-containing antibiotics can use OprO but also the slightly narrower OprP as entries ports into P. aeruginosa. In addition to these studies on OprP and OprO, several other studies were performed connected to the transport through bacterial nanopores. For example, one study looked at the effect of polarizable force fields for the permeation through nanopores, but it had to be concluded that those polarizable force fields were not yet accurate enough at this point in time.
Projektbezogene Publikationen (Auswahl)
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Fosfomycin Permeation through the Outer Membrane Porin OmpF. Biophysical Journal, 116(2), 258-269.
Golla, Vinaya Kumar; Sans-Serramitjana, Eulàlia; Pothula, Karunakar Reddy; Benier, Lorraine; Bafna, Jayesh Arun; Winterhalter, Mathias & Kleinekathöfer, Ulrich
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Computational Modeling of Ion Transport in Bulk and through a Nanopore Using the Drude Polarizable Force Field. Journal of Chemical Information and Modeling, 60(6), 3188-3203.
Prajapati, Jigneshkumar Dahyabhai; Mele, Crystal; Aksoyoglu, Mehmet Alphan; Winterhalter, Mathias & Kleinekathöfer, Ulrich
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Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores. Chemical Science, 11(38), 10344-10353.
Wang, Jiajun; Prajapati, Jigneshkumar Dahyabhai; Kleinekathöfer, Ulrich & Winterhalter, Mathias
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Exploration of Free Energy Surfaces Across a Membrane Channel Using Metadynamics and Umbrella Sampling. Journal of Chemical Theory and Computation, 16(4), 2751-2765.
Golla, Vinaya Kumar; Prajapati, Jigneshkumar Dahyabhai; Joshi, Manas & Kleinekathöfer, Ulrich
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Voltage-Dependent Transport of Neutral Solutes through Nanopores: A Molecular View. The Journal of Physical Chemistry B, 124(47), 10718-10731.
Prajapati, Jigneshkumar Dahyabhai & Kleinekathöfer, Ulrich
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Clostridium perfringens Beta2 toxin forms highly cation-selective channels in lipid bilayers. European Biophysics Journal, 51(1), 15-27.
Benz, Roland; Piselli, Claudio; Hoxha, Cezarela; Koy, Cornelia; Glocker, Michael O. & Popoff, Michel R.
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Fosmidomycin transport through the phosphate‐specific porins OprO and OprP of Pseudomonas aeruginosa. Molecular Microbiology, 116(1), 97-108.
Piselli, Claudio & Benz, Roland
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How to Enter a Bacterium: Bacterial Porins and the Permeation of Antibiotics. Chemical Reviews, 121(9), 5158-5192.
Prajapati, Jigneshkumar Dahyabhai; Kleinekathöfer, Ulrich & Winterhalter, Mathias
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Large‐Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca**. Angewandte Chemie International Edition, 60(15), 8089-8094.
Pangeni, Sushil; Prajapati, Jigneshkumar Dahyabhai; Bafna, Jayesh; Nilam, Mohamed; Nau, Werner M.; Kleinekathöfer, Ulrich & Winterhalter, Mathias
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Cell wall channels of Rhodococcus species: identification and characterization of the cell wall channels of Rhodococcus corynebacteroides and Rhodococcus ruber. European Biophysics Journal, 51(4-5), 309-323.
Piselli, Claudio; Benier, Lorraine; Koy, Cornelia; Glocker, Michael O. & Benz, Roland
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Permeation of Fosfomycin through the Phosphate-Specific Channels OprP and OprO of Pseudomonas aeruginosa. The Journal of Physical Chemistry B, 126(7), 1388-1403.
Golla, Vinaya Kumar; Piselli, Claudio; Kleinekathöfer, Ulrich & Benz, Roland
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Importance of the lysine cluster in the translocation of anions through the pyrophosphate specific channel OprO. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1865(2), 184086.
Piselli, Claudio; Golla, Vinaya Kumar; Benz, Roland & Kleinekathöfer, Ulrich
