Assembly of the MexAB-OprM Multidrug Efflux System of Pseudomonas aeruginosa : Identification and Characterization of Mutations in mexA Compromising MexA Multimerization and Interaction with MexB

Abstract

The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P. aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N-and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobials (21). The resistance has historically been attributed to the presence in this organism of an outer membrane (OM) of low permeability (55), but it is increasingly clear that resistance owes much to the operation of broadly specific, so-called multidrug efflux systems (58-60, 64) that work synergistically with limited OM permeability (18,40,60). Several multidrug efflux systems in P. aeruginosa have been described to date (61), although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (38). Hyperexpression of this system also occurs in so-called nalB (27, 28, 75, 88)-and nalC (75, 88)-type multidrug-resistant mutants. MexAB-OprM accommodates a broad range of structurally diverse antimicrobials, including dyes, detergents, inhibitors of fatty acid biosynthesis, organic solvents, disinfectants, and clinically relevant antibiotics (10,34,37,39,41,42,48,70,74,74,76), and is implicated in the export of homoserine lactones involved in quorum sensing (17, 57) and, possibly, virulence factors (22).The MexAB-OprM efflux system, like the other tripartite Mex efflux systems in P. aeruginosa, consists of an inner membrane (IM) drug-proton antiporter of the resistance-nodulation-cell division (RND) family (MexB), an OM channel-forming component (OprM; also called OM factor [OMF]), and a periplasmic membrane fusion protein (MFP) (MexA) (58, 86). Crystal structures have not yet been reported for any of the efflux components of P. aeruginosa, although structures are available for the homologous OM (TolC [35]) and RND (AcrB [52]) components of the Mex-like AcrAB-TolC multidrug efflux system of Escherichia coli. The TolC channel is a trimer and spans both the OM (as a ␤-barrel) and periplasm (as a ␣-helical barrel) (35). Measuri...

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…Crystal structures of the MFP MexA reveal an elongated monomer with an extended ␣-helical coiled-coil domain and two globular domains consisting primarily of ␤-strands (2,199). Substantial biochemical evidence suggests that MFPs exist as oligomers (329,334,549), and in crystals, MexA protomers oligomerize in spirals, forming the basis for the model in Fig. 14 (blue section).…”

“…In contrast to earlier predictions that suggested that MFPs might span the periplasm with their N and C termini bound to the inner and outer membranes, respectively (548), the structures suggest that both the N and C termini are likely to lie in close proximity to the inner membrane. Mutagenesis and suppressor analyses suggest that the C termini of the MFPs interact with the inner membrane transporters (at least in the case of the RND transporters Mex and Acr) (139,329,330), while the N terminus may be involved in oligomerization of the MFPs (329).…”

Influence of Quorum Sensing and Iron on Twitching Motility and Biofilm Formation in Pseudomonas aeruginosa

“…The bacterial strains and plasmids used in this study are listed in Table 1. Routine growth of P. aeruginosa and Escherichia coli was carried out in Luria broth (LB) as before (52), with antibiotic supplementation to maintain plasmids as needed (for pUC18, ampicillin [100 g/ml]; for mini-CTX-lacZ and its derivatives, tetracycline [10 g/ml for E. coli and 75 to 100 g/ml for P. aeruginosa]; for pK18mobsacB and its derivatives, kanamycin [50 g/ml]; for pFLP2, ampicillin [100 g/ml for E. coli] or carbenicillin [150 g/ml for P. aeruginosa]; and for pMMB207 and its derivatives, chloramphenicol [10 g/ml for E. coli and 75 to 100 g/ml for P. aeruginosa]). Fe-limited BM2 succinate and BM2 glucose minimal media have been described previously (61) and were supplemented with FeCl 3 as and where indicated.…”

Citrate-mediated iron uptake in Pseudomonas aeruginosa: involvement of the citrate-inducible FecA receptor and the FeoB ferrous iron transporter

“…Bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were cultivated at 37 uC in Luria (L) broth as before (Nehme et al, 2004) supplemented with antibiotics to maintain plasmids as needed [pEX18Tc, pRK415 and their derivatives: tetracycline (10 mg ml 21 , E. coli; 75 mg ml 21 , P. aeruginosa); pK18MobSacB and its derivatives: kanamycin (50 mg ml 21 , E. coli; 1500 mg ml 21 , P. aeruginosa); pUCP18 and its derivatives: ampicillin (100 mg ml 21 , E. coli) or carbenicillin (800 mg ml 21 , P. aeruginosa); pUT : : mini-Tn5-tet: tetracycline (10 mg ml 21 , E. coli); pCR-Blunt II-TOPO: ampicillin (100 mg ml 21 , E. coli)]. The iron-limited succinate minimal media [here dubbed 'A' (Meyer & Abdallah, 1978) and 'B' (Poole et al, 1990)] have been described previously.…”