Use of Fluorescence Probes to Monitor Function of the Subunit Proteins of the MexA-MexB-OprM Drug Extrusion Machinery inPseudomonas aeruginosa

Abstract

The MexA-MexB-OprM efflux pump of Pseudomonas aeruginosa consists of two inner membrane proteins, MexA and MexB, and one outer membrane protein, OprM. We investigated the role of the components of this drug extrusion system by evaluating the repercussions of deleting these subunit components on the accumulation of several fluorescent probes. Fluorescence intensities of positively charged 2-(4-dimethylaminostyryl)-1-ethylpyridinium and uncharged N-phenyl-1-naphtylamine were 7 and 4 times higher, respectively, in the mutant lacking OprM and 4 and 1.7 times higher, respectively, in the mutants lacking MexA or MexB than in the wild type strain. This order of fluorescence intensity was fully consistent with a previously reported minimum inhibitory concentration of antibiotics such as tetracycline, chloramphenicol, and fluoroquinolones. Ethidium bromide accumulation in all the Mex mutants proceeded at about 5 times faster than the rate in the wild type cells. This result is in accord with the minimum inhibitory concentration of ␤-lactam antibiotics. These results suggest that the fluorescence probes could be successfully used in real time monitoring of the function of the drug extrusion machinery in Gram-negative bacteria. The downhill extrusion kinetics of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, which orients perpendicular to the inner leaflet of the cytoplasmic membrane, from preloaded cells lacking the extrusion pump was preceded by a slow increase in fluorescence intensity, whereas the wild type cell immediately released the dye. This observation was explained by a slow trans-cytoplasmic membrane crossing of intracellular dye in the mutants. These results reflected higher accumulation of the probe in the cytoplasmic membrane in the mutants and strengthened the hypothesis that extrusion of hydrophobic substrate mediated by MexA-MexB-OprM mainly takes place from the interior of the cytoplasmic membrane.Multiple antibiotic resistance in bacteria has been associated with the overexpression of endogenous efflux genes (for reviews see Refs. 1 and 2). In Pseudomonas aeruginosa, active extrusion (3, 4) combined with a tight diffusion barrier at the outer membrane (5, 6) are the main reasons for low specific resistance against diverse antibiotics and other toxic elements (7).Multidrug resistance in P. aeruginosa is now attributed mainly to the overexpression of three sets of operons. Overexpression of the operon mexA-mexB-oprM is characteristic for nalB type multidrug-resistant mutants, (8, 9), whereas overexpression of the operon mexC-mexD-oprJ (10) is responsible for multidrug resistance in nfxB type mutants (11). A third type designated as nfxC has also been associated with a diminished intracellular drug accumulation (12). The genes coding for the proteins involved in the drug extrusion have been characterized recently (13) and named mexE-mexF-oprN.A common characteristic of multiantibiotic-resistant mutants in P. aeruginosa is their broad resistance to quinolones, and hence one method generally used t...

Bioenergetics of the Staphylococcal Multidrug Export Protein QacA

“…Consistent with these assumptions was the observation that the amount of the fluorescent substrate in loaded cells was proportional to the concentration used. Similar kinetic studies of multidrug efflux systems using whole cells have been performed in P. aeruginosa (32) and Mycobacterium smegmatis (33).…”

Viability states of bacteria—Specific mechanisms of selected probes

“…Unspecific transporters were responsible for immediate export of NBDT from E. coli cells (55). Proton motive force dependent transporters of the RND (resistance/nodulation/division) superfamily were supposed to pump NBDT out, because they are known to be involved in toluene resistance (56) or export of fluorescence probes (57). Deactivation of the efflux pumps using the proton motive force inhibitor and protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was necessary to stabilize cellular NBDT fluorescence (55).…”

VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus

“…AcrB (E. coli) and MexB (P. aeruginosa) are known as H + -coupled transporters (Murakami et al, 2002;Ocaktan et al, 1997;Thanassi et al, 1997), and three characteristic charged residues in the transmembrane domains of these two proteins have been suggested to be part of the protontranslocating pathway (Murakami et al, 2002). Since these residues are also conserved in VmeB of V. parahaemolyticus (K342, E346, D407, D408, K937), this protein is also likely to be energized by proton coupling, a conclusion that is supported by the finding, reported above, that Na + levels and fluxes are unrelated to VmeAB-mediated transport.…”