Related Experiment Videos
Regulation of membrane permeability by a two-component regulatory system in Pseudomonas aeruginosa
Yanping Wang1, Unhwan Ha, Lin Zeng
1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610, USA.
Abstract:
Membrane impermeability is the major contributing factor to multidrug resistance in clinical isolates of Pseudomonas aeruginosa. By using laboratory strain PAK, a spontaneous P. aeruginosa mutant (mutant PAK1-3) whose membrane had reduced permeability and which displayed increased levels of resistance to various antibiotics, especially aminoglycosides, was isolated. By complementation of the mutant with a genomic clone library derived from wild-type strain PAK, a novel two-component regulatory system (PprA and PprB) was identified and was found to be able to increase the permeability of the bacterial membrane and render PAK1-3 sensitive to antibiotics. Furthermore, specific phosphorylation of the response regulator (PprB) by histidine kinase (PprA) was observed in vitro, demonstrating that they are cognate two-component regulatory genes. Introduction of a plasmid expressing the pprB gene into randomly chosen clinical isolates (n = 17) resulted in increased sensitivity to aminoglycosides in the majority of isolates (n = 13) tested. This is the first demonstration that P. aeruginosa membrane permeability can be regulated, providing an important clue in the understanding of the mechanism of membrane impermeability-mediated multidrug resistance in P. aeruginosa.
Insights
Researchers identified a novel two-component system, PprA-PprB, that regulates membrane permeability in Pseudomonas aeruginosa. This discovery offers a potential strategy to overcome multidrug resistance in bacterial infections.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Membrane impermeability is a key factor in multidrug resistance (MDR) in clinical Pseudomonas aeruginosa isolates.
- Reduced membrane permeability leads to increased resistance against various antibiotics, particularly aminoglycosides.
Purpose of the Study:
- To identify genetic factors regulating membrane permeability in Pseudomonas aeruginosa.
- To understand the mechanism of membrane impermeability-mediated multidrug resistance.
Main Methods:
- Isolation and characterization of a spontaneous mutant (PAK1-3) with reduced membrane permeability.
- Complementation of the mutant with a genomic library to identify regulatory genes.
- In vitro phosphorylation assays to confirm the interaction between PprA and PprB.
- Introduction of the pprB gene into clinical isolates to assess its effect on antibiotic sensitivity.
Main Results:
- A novel two-component regulatory system, PprA (histidine kinase) and PprB (response regulator), was identified.
- The PprA-PprB system was shown to increase bacterial membrane permeability, restoring antibiotic sensitivity in the mutant.
- In vitro assays confirmed PprA phosphorylates PprB, validating them as cognate partners.
- Expression of pprB in 13 out of 17 clinical isolates increased their sensitivity to aminoglycosides.
Conclusions:
- The PprA-PprB system is the first identified regulator of membrane permeability in Pseudomonas aeruginosa.
- Targeting this system presents a potential therapeutic strategy to combat multidrug resistance in P. aeruginosa infections.