Drug-Induced Conformational Changes in Multidrug Efflux Transporter AcrB from Haemophilus influenzae

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Abstract
In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrBHI). Similar to other RND transporters AcrBHI associates with AcrAHI, the periplasmic membrane fusion protein, and the outer membrane channel TolCHI. Here, we report that AcrABHI confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolCEC) protein. To investigate the intracellular dynamics of AcrABHI, single cysteine mutations were constructed in AcrBHI in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrABHI and in the presence or absence of AcrAHI and TolCEC. We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrBHI.