Transporters belonging to the r esistance- n odulation- d ivision (RND) superfamily of proteins are invariably present in the genomes of Gram-negative bacteria and are largely responsible for the intrinsic antibiotic resistance of these organisms. The numbers of genes encoding RND transporters per genome vary from 1 to 16 and correlate with the environmental versatilities of bacterial species. Pseudomonas aeruginosa strain PAO1, a ubiquitous nosocomial pathogen, possesses 12 RND pumps, which are implicated in the development of clinical multidrug resistance and known to contribute to virulence, quorum sensing, and many other physiological functions.

Transporters of the resistance-nodulation-cell division (RND) superfamily of proteins are the dominant multidrug efflux power of Gram-negative bacteria. The major RND efflux pump of Pseudomonas aeruginosa is MexAB-OprM, in which the inner membrane transporter MexB is responsible for the recognition and binding of compounds. The high importance of this pump in clinical antibiotic resistance made it a subject of intense investigations and a promising target for the discovery of efflux pump inhibitors. This study is focused on a series of peptidomimetic compounds developed as effective inhibitors of MexAB-OprM. We performed multi-copy molecular dynamics simulations, machine-learning (ML) analyses, and site-directed mutagenesis of MexB to investigate interactions of MexB with representatives of efflux avoiders, substrates, and inhibitors. The analysis of both direct and water-mediated protein-ligand interactions revealed characteristic patterns for each class, highlighting significant differences between them. We found that efflux avoiders poorly interact with the access binding site of MexB, and inhibition engages amino acid residues that are not directly involved in binding and transport of substrates. In agreement, machine-learning models selected different residues predictive of MexB substrates and inhibitors. The differences in interactions were further validated by site-directed mutagenesis. We conclude that the substrate translocation and inhibition pathways of MexB split at the interface (between the main putative binding sites) and at the deep binding pocket and that interactions outside of the hydrophobic patch contribute to the inhibition of MexB. This molecular-level information could help in the rational design of new inhibitors and antibiotics less susceptible to the efflux mechanism. IMPORTANCE: Multidrug transporters recognize and expel from cells a broad range of ligands including their own inhibitors. The difference between the substrate translocation and inhibition routes remains unclear. In this study, machine learning and computational and experimental approaches were used to understand dynamics of MexB interactions with its ligands. Our results show that some ligands engage a certain combination of polar and charged residues in MexB binding sites to be effectively expelled into the exit funnel, whereas others engage aromatic and hydrophobic residues that slow down or hinder the next step in the transporter cycle. These findings suggest that all MexB ligands fit into this substrate-inhibitor spectrum depending on their physico-chemical structures and properties.

The ability Gram-negative pathogens have at adapting and protecting themselves against antibiotics has increasingly become a public health threat. Data-driven models identifying molecular properties that correlate with outer membrane (OM) permeation and growth inhibition while avoiding efflux could guide the discovery of novel classes of antibiotics. Here we evaluate 174 molecular descriptors in 1260 antimicrobial compounds and study their correlations with antibacterial activity in Gram-negative Pseudomonas aeruginosa. The descriptors are derived from traditional approaches quantifying the compounds’ intrinsic physicochemical properties, together with, bacterium-specific from ensemble docking of compounds targeting specific MexB binding pockets, and all-atom molecular dynamics simulations in different subregions of the OM model. Using these descriptors and the measured inhibitory concentrations, we design a statistical protocol to identify predictors of OM permeation/inhibition. We find consistent rules across most of our data highlighting the role of the interaction between the compounds and the OM. An implementation of the rules uncovered in our study is shown, and it demonstrates the accuracy of our approach in a set of previously unseen compounds. Our analysis sheds new light on the key properties drug candidates need to effectively permeate/inhibit P. aeruginosa, and opens the gate to similar data-driven studies in other Gram-negative pathogens.

Multidrug efflux is one of the major mechanisms of antibiotic resistance identified in clinical isolates of the human pathogen Acinetobacter baumannii . The multiple antibiotic resistance in this species is often enabled by the overproduction of the tripartite efflux pump AdeABC. In this pump, AdeB is the inner membrane transporter from the resistance-nodulation-division (RND) superfamily of proteins, which is responsible for the recognition and efflux of multiple structurally unrelated compounds. Like other RND transporters, AdeB is a trimeric protein with ligand-binding sites located in the large periplasmic domains. Previous structural studies, however, highlighted the uniqueness of AdeB interactions with ligands. Up to three ligand molecules were bound to one protomer of AdeB, mapping its substrate translocation path. In this study, we introduced single and double substitutions in the identified ligand-binding sites of AdeB. Our results show that the mechanism of substrate translocation by AdeB is different from that of other characterized RND transporters and that the functional interactions between the sites are nonadditive. We identified AdeB mutants with both the loss and the gain of antibiotic susceptibility phenotypes, as well as AdeB mutations making A. baumannii cells overproducing such pump variants even more susceptible to multiple antibiotics than efflux-deficient cells. IMPORTANCE: Multidrug efflux pumps of the resistance-nodulation-division superfamily of proteins are important contributors to various aspects of bacterial physiology and antibiotic resistance. Studies of the best-characterized model transporter AcrB from Escherichia coli suggested that these transporters operate by a functional rotation mechanism in which various substrates bind to at least two different binding sites. This study suggests that the mechanism of AdeB is distinct and that the binding sites in this transporter are functionally linked.

Klebsiella pneumoniae and Escherichia coli cause a broad range of human infections with multidrug-resistant strains presenting serious therapeutic challenges in clinics. The discovery of new antibiotics effective against these pathogens is hindered because of effective drug permeability barriers comprising active efflux pumps acting synergistically with the low-permeability outer membrane. In this study, we characterized these barriers in K. pneumoniae American Type Culture Collection (ATCC) 43816, a hypervirulent strain broadly used in infection models. For this purpose, we constructed an efflux-deficient strain lacking the outer membrane channel TolC, which is required for activities of most resistance-nodulation-division efflux pumps, a hyperporinated strain producing a large non-specific pore in the outer membrane, and a double-compromised strain combining these two features. We next compared the contributions of the permeability barriers of ATCC 43816 to those of the laboratory E. coli BW25113 in antibiotic susceptibilities and persistence and in the intracellular accumulation of fluorescent probes. We identified significant differences between these two related species. Our results show that the outer membranes of K. pneumoniae and E. coli differ in their permeability to fluorescent probes and antibiotics and that K. pneumoniae is more effective in efflux of various compounds. We further found that nutrient-rich and minimal growth media do not affect permeation of all antibiotics in the same way and that the mechanism of antibiotic action and specific physicochemical properties of each compound play a defining role. Likewise, the roles of permeability barriers in the persistence of K. pneumoniae and E. coli vary, depending on the mechanism of antibiotic action, its external concentration, and the affected barrier.

Multidrug efflux transporters are major contributors to the antibiotic resistance of Acinetobacter baumannii in clinical settings. Previous studies showed that these transporters are tightly integrated into the physiology of A. baumannii and have diverse functions. However, for many of the efflux pumps, such functions remain poorly defined. In this study, we characterized two putative drug efflux pumps, AmfAB and AmfCD (Acinetobacter Major Facilitator), that are homologous to EmrAB-like transporters from Escherichia coli and other Gram-negative bacteria. These pumps comprise the Major Facilitator Superfamily (MFS) transporters AmfB and AmfD and the periplasmic membrane fusion proteins AmfA and AmfC, respectively. We inactivated and overproduced these pumps in the wild-type ATCC 17978 strain and its derivative strains lacking the major efflux pumps from the Resistance–Nodulation–Division (RND) superfamily and characterized antibiotic susceptibilities and growth of the strains under stresses typical during human infections. We found that neither AmfAB nor AmfCD contribute to the antibiotic non-susceptibility phenotypes of A. baumannii. The two pumps, however, are critical for the adaptation and growth of the bacterium under acidic stress, whereas AmfCD also contributes to growth under conditions of low iron, high temperature, and in the presence of bile salts. These functions are dependent on the presence of the RND pumps, the inactivation of which further diminishes A. baumannii survival and growth. Our results suggest that MFS transporters contribute to stress survival by affecting the permeability properties of the A. baumannii cell envelope.