Functional and pharmacological characterization of Na+/H+ antiport systems of Klebsiella pneumoniae
Duration: 24 months
Finance source: State budget, contracting authority: Romanian National Authority for Scientific Research and
Innovation, CNCS – UEFISCDI
Budget: 550000 RON
Project start: 01.10.2015
Project end: 30.09.2017
Regulation of intracellular Na+ concentration and pH is a prerequisite for the survival of any organism. The main effectors of this process are secondary active transport systems, the Na+/H+ antiporters or exchangers that exist in virtually all organisms. Understanding of Na+/H+ antiporters is crucial as they have been shown in humans to be involved in various pathologies, while in bacteria they can be essential for survival and virulence. Klebsiella pneumoniae is one pathogen that has, in recent years, become resistant to common antibiotic therapy and can cause infections with a high mortality rate in immunocompromised or debilitated patients. A better understanding of transporter systems in K. pneumoniae might thus allow for development of more effective treatment options. In the framework of our project, we plan to characterize the Na+/H+ transport systems of K. pneumoniae: KpNhaA1, KpNhaA2, KpNhaB and KpNhaP2. This will be done mainly by the use of solid-supported membrane-based electrophysiology in combination with fluorimetric techniques. Overall, our goal is to obtain a better understanding of Na+/H+ antiport in K. pneumoniae and determine kinetic parameters allowing us to model antiporter function under given environmental conditions. We will also test various inhibitors in order to determine potential means of inhibiting Na+/H+ antiport in K. pneumoniae.
Lecturer Dr. Octavian Călinescu – Project Director
Prof. Dr. Constanța Ganea – Senior Researcher
Assistant Prof. Dr. Adrian Iftime – Postdoctoral Researcher
Ioana Tofolean – PhD Student
Alexandru Filippi – PhD Student
Rodica Zaharia – Technical Assistant
1. Călinescu, O., Fendler, K., “A Universal Transport Mechanism for Na+/H+ Exchangers”, International Workshop: Proton and Proton-coupled Transport, 27-29 mai 2016, Berlin, Germany.
2. Călinescu, O., Iftime, A., Ganea, C., Fendler, K., “Electrophysiological Investigation of Prokaryotic Na+/H+ Exchangers”, Conferința Națională de Biofizică, 2-4 iunie 2016, Cluj-Napoca, Romania, ISSN: 2248-0749.
3. Patino-Ruiz, M., Ganea, C., Fendler, K., Călinescu, O., “Functional investigation of Na+/H+ exchangers from the pathogen Klebsiella pneumoniae”, Biophysics and Medicine of Channels and Transporters: Electrifying New Insights, 14.05.2017 – 20.05.2017, Erice, Italy.
1. Călinescu, O., Linder, M., Wöhlert, D., Yildiz, Ö., Kühlbrandt, W., Fendler, K. 2016. Electrogenic Cation Binding in the Electroneutral Na+/H+ Antiporter of Pyrococcus abyssi, J. Biol. Chem. 291, 26786-26793.
2. Călinescu, O., Dwivedi, M., Patino-Ruiz, M., Padan, E., Fendler, K. 2017. Lysine300 Is Essential for Stability but not for Electrogenic Transport of the E. coli NhaA Na+/H+ Antiporter, J. Biol. Chem. 292, 7932-7941.
3. Patino-Ruiz, M., Ganea, C., Fendler, K., Călinescu, O. 2017. Competition is the basis of the transport mechanism of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae. PLOS ONE. 12, e0182293.
- Setting up the equipment for Solid-Supported Membrane (SSM)-based electrophysiology and preliminary measurements (ACHIEVED)
- Over-expression of K. pneumoniae Na+/H+ exchangers KpNhaA1, KpNhaA2, KpNhaB, KpNhaP2 in E. coli (ACHIEVED)
- Testing the functionality of the K. pneumoniae Na+/H+ exchangers (ACHIEVED)
- Solubilization and purification of the K. pneumoniae Na+/H+ exchangers (ACHIEVED)
- Testing the activity of the the K. pneumoniae Na+/H+ exchangers using SSM-based electrophysiology (ACHIEVED)
- Full electrophysiological characterization of KpNhaA1, KpNhaA2 and KpNhaB (ACHIEVED)
- Characterization of KpNhaP2 by electrophysiological or fluorescence measurements (ACHIEVED)
- Evaluating potential inhibitors for the K. pneumoniae Na+/H+ exchangers (ACHIEVED)
- Analysis and kinetic modelling of the K. pneumoniae Na+/H+ exchangers (ACHIEVED)
- Publishing 3 journal articles (ACHIEVED)
Scientific activity reports