Interactions underlying assembly of the Escherichia coli AcrAB–TolC multidrug efflux system

Abstract

SummaryThe major Escherichia coli multidrug efflux pump AcrAB-TolC expels a wide range of antibacterial agents. Using in vivo cross-linking, we show for the first time that the antiporter AcrB and the adaptor AcrA, which form a translocase in the inner membrane, interact with the outer membrane TolC exit duct to form a contiguous proteinaceous complex spanning the bacterial cell envelope. Assembly of the pump appeared to be constitutive, occurring in the presence and absence of drug efflux substrate. This contrasts with substrate-induced assembly of the closely related TolC-dependent protein export machinery, possibly reflecting different assembly dynamics and degrees of substrate responsiveness in the two systems. TolC could be cross-linked independently to AcrB, showing that their large periplasmic domains are in close proximity. However, isothermal titration calorimetry detected no interaction between the purified AcrB and TolC proteins, suggesting that the adaptor protein is required for their stable association in vivo . Confirming this view, AcrA could be cross-linked independently to AcrB and TolC in vivo , and calorimetry demonstrated energetically favourable interactions of AcrA with both AcrB and TolC proteins. AcrB was bound by a polypeptide spanning the C-terminal half of AcrA, but binding to TolC required interaction of N-and C-terminal polypeptides spanning the lipoyl-like domains predicted to present the intervening coiled-coil to the periplasmic coils of TolC. These in vivo and in vitro analyses establish the central role of the AcrA adaptor in drug-independent assembly of the tripartite drug efflux pump, specifically in coupling the inner membrane transporter and the outer membrane exit duct.

Structure of the periplasmic component of a bacterial drug efflux pump

“…The structure shows that residues 29 and 259 of MexA lie within 5 Å of each other in the ␤-barrel domain, positioning the C terminus in close proximity to the N terminus, not near the outer membrane. Experimental evidence that the C-terminal domain contacts the inner membrane components (12,22) also excludes it from interaction with the outer membrane. Furthermore, ␣-helical hairpin formation would need to occur reversibly, with the two long ␣-helices interacting to draw together the inner and outer membranes.…”

“…Nevertheless, genetic analysis shows that assembly of functional drug efflux and protein export pumps requires the third component, the adaptor protein (6,11). Indeed, direct interactions between all three components of efflux pumps have been detected by cross-linking studies, in both protein export (5) and antibiotic efflux systems (12).…”

Insights into virulence factors determining the pathogenicity ofCronobacter sakazakii

“…86 Interestingly, ibeB (a gene synonymous with cusC) in C. sakazakii has been reported, belonging to constellate of genes encoding a copper and silver resistance cation efflux system, ultimately allowing the invasion to brain microvascular endothelial cells (BMEC) cells. 23, 87 When assessed by Kucerova et al 23 it was discovered that the entire cation efflux operon (cusA, cusB and cusC) and its regulatory gene cusR were present in isolates colligated with neonatal infections (including C. sakazakii ATCC 29544 T , 696, 701, 767, C. malonaticus and C. turicensis) and absent in the other strains evaluated (C. sakazakii B894, ATCC 12868, 20, C. dublinensis and C. muytjensii).…”

A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps

“…They are, therefore, important to bacterial survival, especially during infections. In all cases, an outer membrane (OM)-anchored TolC exit duct is recruited by a substrate-loaded inner membrane (IM) translocase, comprising a proton antiporter or ATPase, and an adaptor protein, establishing tripartite exit portals that span both cell membranes and the intervening periplasmic space (6)(7)(8)(9). Tripartite protein export and drug efflux machineries are active only when all three components are present.…”