Two novel outer membrane proteins involved in intrinsic aminoglycoside resistence in Pseudomonas aeruginosa

The expression of tripartite multi-drug efflux pumps such as MexA-MexB-OprM in Pseudomonas aeruginosa contributes to intrinsic resistance to a wide variety of antimicrobials, including (3-lactams, chloramphenicol, macrolides, quinolones, and tetracycline. MexX-MexY are the only linker and pump efflux system components in P. aeruginosa that have been shown to confer intrinsic resistance to aminoglycosides. While a number of studies suggest that OprM, the main efflux outer membrane protein (OMP), forms a functional channel with the MexX-MexY proteins, other data suggests that another OMP is the native channel for the MexX-MexY efflux system. Fifteen functionally uncharacterized OprM-homologues identified in the recently-sequenced genome of P. aeruginosa were possible candidates for the role of the native outer membrane channel for MexX-MexY. Insertional inactivation of OpmG resulted in an 8-fold decrease in MIC to streptomycin, kanamycin, and gentamicin, while inactivation of OpmH resulted in 4- to 8-fold decreases in MIC to kanamycin and streptomycin. When reintroduced into P. aeruginosa on multicopy plasmids, both OpmG and OpmH were able to complement the susceptibility of their respective mutants. Changes in MIC due to pseudo-reversion through compensatory mutations were not a factor, as demonstrated by mini-microarray hybridization analysis of the OprM-homologues. This study demonstrates that the two novel outer membrane proteins OpmG and Opmh play a role in aminoglycoside resistance, and that OpmG is likely the main aminoglycoside efflux channel.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat