Non‐equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump <scp>TriABC</scp> from <scp><i>P</i></scp><i>seudomonas aeruginosa</i>

Weeks, Jon W.; Nickels, Logan M.; Ntreh, Abigail T.; Zgurskaya, Helen I.

doi:10.1111/mmi.13124

Cited by 19 publications

(19 citation statements)

References 35 publications

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“…In the asymmetric MFP dimer TriAB, these two functions were previously assigned to TriB and TriA, respectively (11). In this study, site-specific mutagenesis and disulfide cross-linking experiments showed that TriB predominantly binds to the H4-H7 interprotomer groove of OpmH and promotes opening of the channel, thus coupling the opening of the channel to the stimulation of the transporter.…”

Section: Discussionmentioning

confidence: 84%

“…To confirm the functionality of OpmH His , JWW9 (attTn7:: P BAD -opmH His ⌬ompH::Gm) cells were transformed with the pBSPII vector and its derivatives pPS1824 and pBSPII-AxBC, which express the native TriABC pump and the covalently linked TriAxB protein along with TriC, respectively, from the constitutive P lac promoter (11). A covalent linkage between TriA and TriB ensures that the proteins are produced in equal quantities and assemble stoichiometric complexes with TriC and OpmH.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to AcrAB-TolC, in which a single MFP, AcrA, is responsible for the assembly of the complex and opening of the channel, this transporter requires two different MFPs, TriA and TriB, that play nonequivalent roles in the pump's function (11,12). Our previous results suggested that TriA is responsible for the recruitment of the OMF, while TriB is responsible for stimulation of the transporter TriC (11). Here, we used the functional and structural differences between the two MFPs to link their functional roles to specific interactions with the OMF OpmH.…”

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confidence: 99%

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

et al. 2016

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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. IMPORTANCEMultidrug 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. In Gram-negative bacteria, polyspecific transporters detoxify the inner membrane and periplasm of noxious compounds and contribute to clinical antibiotic resistance (1). A characteristic structural feature of these transporters is the formation of tripartite complexes spanning both the inner and outer membranes of Gram-negative cell envelopes. Located in the outer membrane are proteins belonging to the outer membrane factor (OMF) family that act as channels for substrate expulsion across the low-permeability barrier of the outer membrane. OMFs show very little sequence similarity to one another but are structurally similar (2). OMF structures comprise both a ␤-barrel domain, a common structural feature of other outer membrane proteins, and a large ␣-helical barrel (3) (Fig. 1A). The periplasmic tip of the ␣-helical barrel converges to form a closed aperture that prevents leakage of the periplasmic content and uptake of various molecules from the external medium (Fig. 1B). Large extracellular loops on the external side of the ␤-barrel domain also help with blocking noxious chemicals from entering the cell. At the aperture, a series of ionic bridges act as the locking gate of the aperture and disruption of these causes a "leaky" phenotype (4, 5). A secondary gate of aspartate or, in a few cases, a ring of hydrophobic residues acts additionally to keep the aperture locked.In the resting state,...

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Section: Discussionmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 2016

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“…Recently, it has been proposed that some bacterial efflux pumps require two different PAPs for functional activity, namely the TriABC-OpmH triclosan extrusion system of Pseudomonas aeruginosa and the ZrpADBC pump of Serratia sp. ATCC 39006 (Mima et al, 2007;Gristwood et al, 2008;Weeks et al, 2015). Alternatively, it is possible that Sll0141 and Sll0180 are active in yet undescribed mechanisms that indirectly affect the T1SS.…”

Section: Tolc-mediated Protein Secretionmentioning

confidence: 99%

Identification of inner membrane translocase components of TolC‐mediated secretion in the cyanobacterium Synechocystis sp. PCC 6803

Gonçalves

Pacheco

Tamagnini

et al. 2018

Environmental Microbiology

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Cyanobacteria were the first organisms ever to perform oxygenic photosynthesis and still significantly contribute to primary production on a global scale. To assure the proper functioning of their primary metabolism and cell homeostasis, cyanobacteria must rely on efficient transport systems to cross their multilayered cell envelope. However, cyanobacterial secretion mechanisms remain largely unknown. Here, we report on the identification of 11 putative inner membrane translocase components of TolC-mediated secretion in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Gene-inactivation of each of the candidate genes followed by a comprehensive phenotypic characterization allowed to link specific protein components to the processes of protein export (as part of the type I secretion system) and drug efflux (part of the resistance-division-nodulation efflux pumps). In addition, mutants in genes sll0141, sll0180 and slr0369 exhibited alterations in pilin glycosylation, but pili structures could still be observed by transmission electron microscopy. By studying the release of outer membrane vesicles (OMVs), an alternative secretion route, on mutants with impaired secretory functions we suggest that the hyper-vesiculating phenotype of the TolC-deficient mutant is related to cell envelope stress management. Altogether, these findings highlight how both classical (TolC-mediated) and nonclassical (OMVs-mediated) secretion systems are crucial for cyanobacterial cell homeostasis.

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“…14 One protomer in each dimer is thought to be responsible for the stimulation of transporter turnover, whereas the second protomer is important for functional interactions with TolC (Figure 6). Hence, the oligomerization of AcrA is essential not only for the assembly of the trans-periplasmic drug tunnel but also for the coupling of transport processes in the two different membranes.…”

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confidence: 99%

Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

et al. 2016

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Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for the development of effective EPIs, especially in light of constantly emerging resistance. Here, we describe EPIs that interact with periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes, and potentiate the activities of antibiotics in Escherichia coli and other Gram-negative bacteria. Our findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.

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

Cited by 19 publications

References 35 publications

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

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

Identification of inner membrane translocase components of TolC‐mediated secretion in the cyanobacterium Synechocystis sp. PCC 6803

Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

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