Pyridylpiperazine-based allosteric inhibitors of RND-type multidrug efflux pumps

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

Resistance-Nodulation-Cell Division (RND) Transporter AcrD Confers Resistance to Egg White in Salmonella enterica Serovar Enteritidis

The excellent survival ability of Salmonella enterica serovar Enteritidis (S. Enteritidis) in egg white leads to outbreaks of salmonellosis frequently associated with eggs and egg products. Our previous proteomic study showed that the expression of multidrug efflux RND transporter AcrD in S. Enteritidis was significantly up-regulated (4.06-fold) in response to an egg white environment. In this study, the potential role of AcrD in the resistance of S. Enteritidis to egg white was explored by gene deletion, survival ability test, morphological observation, Caco-2 cell adhesion and invasion. It was found that deletion of acrD had no apparent effect on the growth of S. Enteritidis in Luria-Bertani (LB) broth but resulted in a significant (p < 0.05) decrease in resistance of S. Enteritidis to egg white and a small number of cell lysis. Compared to the wild type, a 2-log population reduction was noticed in the ΔacrD mutant with different initial concentrations after incubation with egg white for 3 days. Furthermore, no significant difference (p > 0.05) in the adhesion and invasion was found between the wild type and ΔacrD mutant in LB broth and egg white, but the invasion ability of the ΔacrD mutant in egg white was significantly (p < 0.05) lower than that in LB broth. This indicates that acrD is involved in virulence in Salmonella. Taken together, these results reveal the importance of AcrD on the resistance of S. Enteritidis to egg white.

Dilated cardiomyopathy associated with a mutation in the dispatched RND transporter family member 1 gene

Dilated cardiomyopathy is the most common presentation of cardiomyopathy in children with 20–35% of patients having an identified genetic component. There are more than 30 genes implicated in the pathogenesis of dilated cardiomyopathy. We present the first report of a female infant with dilated cardiomyopathy with a genetic variant in the dispatched RND transporter family member 1 gene.

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.ImportanceAntibiotic 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.

Dispatching plasma membrane cholesterol and Sonic Hedgehog dispatch: two sides of the same coin?

Vertebrate and invertebrate Hedgehog (Hh) morphogens signal over short and long distances to direct cell fate decisions during development and to maintain tissue homeostasis after birth. One of the most important questions in Hh biology is how such Hh signaling to distant target cells is achieved, because all Hh proteins are secreted as dually lipidated proteins that firmly tether to the outer plasma membrane leaflet of their producing cells. There, Hhs multimerize into light microscopically visible storage platforms that recruit factors required for their regulated release. One such recruited release factor is the soluble glycoprotein Scube2 (Signal sequence, cubulin domain, epidermal-growth-factor-like protein 2), and maximal Scube2 function requires concomitant activity of the resistance-nodulation-division (RND) transporter Dispatched (Disp) at the plasma membrane of Hh-producing cells. Although recently published cryo-electron microscopy-derived structures suggest possible direct modes of Scube2/Disp-regulated Hh release, the mechanism of Disp-mediated Hh deployment is still not fully understood. In this review, we discuss suggested direct modes of Disp-dependent Hh deployment and relate them to the structural similarities between Disp and the related RND transporters Patched (Ptc) and Niemann-Pick type C protein 1. We then discuss open questions and perspectives that derive from these structural similarities, with particular focus on new findings that suggest shared small molecule transporter functions of Disp to deplete the plasma membrane of cholesterol and to modulate Hh release in an indirect manner.

Bacterial Metal Resistance: Coping with Copper without Cooperativity?

In Escherichia coli and other Gram-negative bacteria, tripartite efflux pumps (TEPs) span the entire cell envelope and serve to remove noxious molecules from the cell. CusBCA is a TEP responsible for copper and silver detoxification in E. coli powered by the resistance-nodulation-cell division (RND) transporter, CusA.

Interchangeability of Periplasmic Adaptor Proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella Typhimurium

Background 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 into 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 E. 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.

Interactions of a bacterial RND transporter with a transmembrane small protein in a lipid environment

The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases the resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that the drug sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate the activity of AcrB. This mode of regulation by a small protein and lipid may occur for other membrane proteins.

Efflux-Mediated Resistance to New Oxazolidinones and Pleuromutilin Derivatives inEscherichia coliwith Class Specificities in the Resistance-Nodulation-Cell Division-Type Drug Transport Pathways

ABSTRACTA major contribution of the resistance-nodulation-cell division (RND)-transporter AcrB to resistance to oxazolidinones and pleuromutilin derivatives inEscherichia coliwas confirmed. However, we discovered significant differences in efflux inhibitor activities, specificities of the homologous pump YhiV (MdtF), and the impact of AcrB pathway mutations. Particularly, entrance channel double-mutation I38F I671T and distal binding pocket mutation F615A revealed class-specific transport routes of oxazolidinones and pleuromutilin derivatives. The findings could contribute to the understanding of the RND-type multidrug transport pathways.