Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC.

TriABC-OpmH is an efflux pump from Pseudomonas aeruginosa with an unusual substrate specificity and protein composition. When overexpressed, this pump confers a high level of resistance to the biocide triclosan and the detergent SDS, which are commonly used in combinations for antimicrobial treatments. This activity requires an RND transporter (TriC), an outer membrane channel (OpmH), and two periplasmic membrane fusion proteins (TriA and TriB) with nonequivalent functions. In the active complex, TriA is responsible for the recruitment of OpmH, while TriB is responsible for stimulation of the transporter TriC. Here, we used the functional and structural differences between the two membrane fusion proteins to link their functional roles to specific interactions with OpmH. Our results provide evidence that the TriB-dependent stimulation of the TriC transporter is coupled to opening of the OpmH aperture through binding to the interprotomer groove of OpmH. IMPORTANCE: Multidrug efflux transporters are important contributors to intrinsic and acquired antibiotic resistance in clinics. In Gram-negative bacteria, these transporters have a characteristic tripartite architecture spanning the entire two-membrane cell envelope. How such complexes are assembled and how the reactions separated in two different membranes are coupled to provide efficient efflux of various compounds across the cell envelope remain unclear. This study addressed these questions, and the results suggest a mechanism for functional integration of drug efflux by the inner membrane transporter and opening of the channel for transport across the outer membrane.

Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa.

In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.

A "Drug Sweeping" State of the TriABC Triclosan Efflux Pump from Pseudomonas aeruginosa.

The structure of the TriABC inner membrane component of the triclosan/SDS-specific efflux pump from Pseudomonas aeruginosa was determined by cryoelectron microscopy to 4.5 Å resolution. The complete structure of the inner membrane transporter TriC of the resistance-nodulation-division (RND) superfamily was solved, including a partial structure of the fused periplasmic membrane fusion subunits, TriA and TriB. The substrate-free conformation of TriABC represents an intermediate step in efflux complex assembly before the engagement of the outer membrane channel. Structural analysis identified a tunnel network whose constriction impedes substrate efflux, indicating inhibition of TriABC in the unengaged state. Blind docking studies revealed binding to TriC at the same loci by substrates and bulkier non-substrates. Together with functional analyses, we propose that selective substrate translocation involves conformational gating at the tunnel narrowing that, together with conformational ordering of TriA and TriB, creates an engaged state capable of mediating substrate efflux.

Mechanism of coupling drug transport reactions located in two different membranes.

Gram- negative bacteria utilize a diverse array of multidrug transporters to pump toxic compounds out of the cell. Some transporters, together with periplasmic membrane fusion proteins (MFPs) and outer membrane channels, assemble trans-envelope complexes that expel multiple antibiotics across outer membranes of Gram-negative bacteria and into the external medium. Others further potentiate this efflux by pumping drugs across the inner membrane into the periplasm. Together these transporters create a powerful network of efflux that protects bacteria against a broad range of antimicrobial agents. This review is focused on the mechanism of coupling transport reactions located in two different membranes of Gram-negative bacteria. Using a combination of biochemical, genetic and biophysical approaches we have reconstructed the sequence of events leading to the assembly of trans-envelope drug efflux complexes and characterized the roles of periplasmic and outer membrane proteins in this process. Our recent data suggest a critical step in the activation of intermembrane efflux pumps, which is controlled by MFPs. We propose that the reaction cycles of transporters are tightly coupled to the assembly of the trans-envelope complexes. Transporters and MFPs exist in the inner membrane as dormant complexes. The activation of complexes is triggered by MFP binding to the outer membrane channel, which leads to a conformational change in the membrane proximal domain of MFP needed for stimulation of transporters. The activated MFP-transporter complex engages the outer membrane channel to expel substrates across the outer membrane. The recruitment of the channel is likely triggered by binding of effectors (substrates) to MFP or MFP-transporter complexes. This model together with recent structural and functional advances in the field of drug efflux provides a fairly detailed understanding of the mechanism of drug efflux across the two membranes.