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Subunit swapping in the Mex-extrusion pumps in Pseudomonas aeruginosa
H Yoneyama1, A Ocaktan, N Gotoh
1Department of Molecular Life Science, School of Medicine, Tokai University, Isehara, Japan.
Abstract:
Pseudomonas aeruginosa encodes three sets of antibiotic extrusion proteins designated as MexA,B.OprM, MexC,D-OprJ and MexE,F-OprN regulated by the nalB, nfxB and nfxC genes, respectively. MexB,D,F, OprM, J,N and MexA,C,E function as the inner membrane pumps, the outer membrane channels and the membrane fusion proteins, respectively. To investigate the possibility of subunit interchangeability, we constructed the following combinations of chimeric pumps: MexA,D-OprM/delta MexB, MexC,B-OprM/delta MexA, and MexA,B-OprJ/delta OprM. The strains producing MexA,D-OprM/delta MexB and MexC,B-OprM/delta MexA failed to restore the antibiotic resistance shown in the strains producing the natural combinations of the subunit proteins. These results suggested that the inner membrane components cannot be interchanged. In contrast, the stains producing MexA,B-OprJ/delta OprM exhibited higher resistance to several antibiotics than the mutant lacking OprM and lower resistance than the strain overexpressing OprM. This result suggests that OprJ may complement the OprM function partially. A spectrum of antibiotics, of which the minimum inhibitory concentrations were restored partially by the complementation, was the same as the spectrum to which the nalB type mutant shows resistance. We surmised from these results that the MexA/MexB unit sustains the substrate specificity of the MexA,B-OprM machinery.
Insights
Investigating Pseudomonas aeruginosa's antibiotic resistance, this study found inner membrane pump components are not interchangeable. However, outer membrane channel OprJ partially complements OprM function, suggesting the MexA/MexB unit dictates substrate specificity.
Area of Science:
- Microbiology
- Molecular Biology
- Drug Resistance
Background:
- Pseudomonas aeruginosa possesses three major multidrug efflux pump systems: MexA,B-OprM, MexC,D-OprJ, and MexE,F-OprN.
- These systems are regulated by nalB, nfxB, and nfxC genes, respectively.
- Each system comprises inner membrane (IM) pumps, outer membrane (OM) channels, and membrane fusion proteins (MFPs).
Purpose of the Study:
- To explore the interchangeability of subunits within the Mex efflux pump systems.
- To determine if heterologous combinations of IM pumps, OM channels, and MFPs can restore antibiotic resistance.
- To elucidate the role of specific subunits in substrate specificity and pump function.
Main Methods:
- Construction of chimeric multidrug efflux pumps by combining subunits from different Mex systems.
- Generation of Pseudomonas aeruginosa strains expressing these chimeric pumps.
- Assessment of antibiotic resistance profiles, including minimum inhibitory concentrations (MICs), for engineered strains.
Main Results:
- Chimeric pumps with interchanged inner membrane components (MexB, MexD, MexF) failed to restore antibiotic resistance.
- A chimeric pump combining MexA/MexB with OprJ (an outer membrane channel) showed partial restoration of antibiotic resistance.
- The antibiotic spectrum affected by OprJ complementation matched that of nalB-type mutants, indicating partial functional overlap.
Conclusions:
- The inner membrane pump components of Pseudomonas aeruginosa's Mex efflux systems are not functionally interchangeable.
- The outer membrane channel OprJ can partially substitute for OprM, suggesting some functional redundancy.
- The MexA/MexB unit appears to be a key determinant of substrate specificity for the MexA,B-OprM efflux machinery.