Mutations in the TolC periplasmic domain affect substrate specificity of the AcrAB-TolC pump

TolC and the other members of the outer membrane factor (OMF) family are outer membrane proteins forming trimeric channels that serve as a conduit for most actively effluxed substrates in Gram-negative bacteria by providing a key component in a multitude of tripartite efflux-pumps. Current models of tripartite pump assembly ascribe substrate selection to the inner-membrane transporter and periplasmic-adapter protein (PAP) assembly, suggesting that TolC is a passive, non-selective channel. While the membrane-embedded portion of the protein adopts a porin-like fold, the periplasmic domain of TolC presents a unique “alpha-barrel” architecture. This alpha-barrel consists of pseudo-continuous α-helices forming curved coiled-coils, whose tips form α-helical hairpins, relaxation of which results in a transition of TolC from a closed to an open-aperture state allowing effective efflux of substrates through its channel. Here, we analysed the effects of site-directed mutations targeting the alpha-barrel of TolC, of the principal tripartite efflux-pump Escherichia coli AcrAB-TolC, on the activity and specificity of efflux. Live-cell functional assays with these TolC mutants revealed that positions both at the periplasmic tip of, and partway up the TolC coiled-coil alpha-barrel domain are involved in determining the functionality of the complex. We report that mutations affecting the electrostatic properties of the channel, particularly the D371V mutation, significantly impact growth even in the absence of antibiotics, causing hyper-susceptibility to all tested efflux-substrates. These results suggest that inhibition of TolC functionality is less well-tolerated than deletion of tolC, and such inhibition may have an antibacterial effect. Significantly and unexpectedly, we identified antibiotic-specific phenotypes associated with novel TolC mutations, suggesting that substrate specificity may not be determined solely by the transporter protein or the PAP, but may reside at least partially with the TolC-channel. Furthermore, some of the effects of mutations are difficult to reconcile with the currently prevalent tip-to-tip model of PAP-TolC interaction due to their location higher-up on the TolC alpha-barrel relative to the proposed PAP-docking sites. Taken together our results suggest a possible new role for TolC in vetting of efflux substrates, alongside its established role in tripartite complex assembly

Tripartite efflux pumps: energy is required for dissociation, but not assembly or opening of the outer membrane channel of the pump

The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE-E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy-independent. Cells expressing wild-type MtrD, in an MtrCE background, were vancomycin-insensitive, but moderately sensitive in an MtrCE-E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE-E434K vancomycin-insensitive; imply that the pump-component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy-dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE-E434K is unable to do so, resulting in the vancomycin-sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy-independent, while both drug export and complex dissociation require active proton flux

The membrane-proximal domain of the periplasmic adapter protein plays a role in vetting substrates utilising channels 1 and 2 of RND efflux transporters

Active efflux by resistance-nodulation-division (RND) efflux pumps is a major contributor to antibiotic resistance in clinically relevant Gram-negative bacteria. Tripartite RND pumps, such as AcrAB-TolC of Salmonella enterica serovar Typhimurium, comprise of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. Previously, we elucidated binding sites within the PAP AcrA (termed binding boxes) that were important for AcrB-transporter recognition. Here, we have refined the binding box model by identifying the most critical residues involved in PAP-RND binding and show that the corresponding RND-binding residues in the closely related PAP AcrE are also important for AcrB interactions. In addition, our analysis identified a membrane-proximal domain (MPD)-residue in AcrA (K366), that when mutated, differentially affects transport of substrates utilising different AcrB efflux-channels, namely channels 1 and 2, supporting a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket (PBP) of the transporter and substrate vetting. Our model predicts that there is a close interplay between the MPD of the PAP and the RND transporter in the productive export of substrates utilising the PBP. Importance Antibiotic resistance greatly threatens our ability to treat infectious diseases. In Gram-negative bacteria, overexpression of tripartite efflux pumps, such as AcrAB-TolC, contributes to multidrug resistance because they export many different classes of antibiotics. The AcrAB-TolC pump is made up of three components: the periplasmic adaptor protein (PAP) AcrA, the RND-transporter AcrB, and the outer-membrane factor TolC. Here, we identified critical residues of AcrA that are important for its function with AcrB in Salmonella enterica serovar Typhimurium. Also, we show that AcrA shares these critical residues with AcrE, a closely related PAP, explaining their interoperability with AcrB. Importantly, we identified a residue in the membrane-proximal domain of AcrA that when mutated affected how different substrates access AcrB and impacted downstream efflux via TolC channel. Understanding the role that PAPs play in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance

Modular design of the selectivity filter pore loop in a novel family of prokaryotic inward rectifier' (NirBac) channels

Potassium channels exhibit a modular design with distinct structural and functional domains; in particular, a highly conserved pore-loop sequence that determines their ionic selectivity. We now report the functional characterisation of a novel group of functionally non-selective members of the prokaryotic inward rectifier' subfamily of K + channels. These channels share all the key structural domains of eukaryotic and prokaryotic Kir/KirBac channels, but instead possess unique pore-loop selectivity filter sequences unrelated to any other known ionic selectivity filter. The strikingly unusual architecture of these NirBac' channels defines a new family of functionally non-selective ion channels, and also provides important insights into the modular design of ion channels, as well as the evolution of ionic selectivity within this superfamily of tetrameric cation channels

Interchangeability of periplasmic adaptor proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella enterica serovar Typhimurium

Background Resistance-nodulation-division (RND) efflux pumps are important mediators of antibiotic resistance. RND pumps, including the principal multidrug efflux pump AcrAB-TolC in Salmonella, are tripartite systems with an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB binding are also involved in AcrD binding. Methods The acrD and acrE genes were expressed in a strain lacking acrABDEF (Δ3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined. Results Co-expression of acrD and acrE significantly decreased susceptibility of the Δ3RND strain to AcrD substrates, showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of Escherichia coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates. Conclusions These data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition

Structural and functional analysis of the Escherichia coli acid-sensing histidine kinase EvgS

The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this

A role for the periplasmic adaptor protein AcrA in vetting substrate access to the RND efflux transporter AcrB

Tripartite resistance-nodulation-division (RND) efflux pumps, such as AcrAB-TolC of Salmonella Typhimurium, contribute to antibiotic resistance and comprise an inner membrane RND-transporter, an outer membrane factor, and a periplasmic adaptor protein (PAP). The role of the PAP in the assembly and active transport process remains poorly understood. Here, we identify the functionally critical residues involved in PAP-RND-transporter binding between AcrA and AcrB and show that the corresponding RND-binding residues in the closely related PAP AcrE, are also important for its interaction with AcrB. We also report a residue in the membrane-proximal domain of AcrA, that when mutated, differentially affects the transport of substrates utilising different AcrB efflux channels, namely channels 1 and 2. This supports a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket of the transporter and substrate vetting. Understanding the PAP’s role in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency