Resistance-nodulation-cell Division Transporter Research Articles

Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Common recognition topology of mex transporters of Pseudomonas aeruginosa revealed by molecular modelling.

The secondary transporters of the resistance-nodulation-cell division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria like Pseudomonas aeruginosa. Among these RND transporters, MexB, MexF, and MexY, with partly overlapping specificities, have been implicated in pathogenicity. Only the structure of the former has been resolved experimentally, which together with the lack of data about the functional dynamics of the full set of transporters, limited a systematic investigation of the molecular determinants defining their peculiar and shared features. In a previous work (Ramaswamy et al., Front. Microbiol., 2018, 9, 1144), we compared at an atomistic level the two main putative recognition sites (named access and deep binding pockets) of MexB and MexY. In this work, we expand the comparison by performing extended molecular dynamics (MD) simulations of these transporters and the pathologically relevant transporter MexF. We employed a more realistic model of the inner phospholipid membrane of P. aeruginosa and more accurate force-fields. To elucidate structure/dynamics-activity relationships we performed physico-chemical analyses and mapped the binding propensities of several organic probes on all transporters. Our data revealed the presence, also in MexF, of a few multifunctional sites at locations equivalent to the access and deep binding pockets detected in MexB. Furthermore, we report for the first time about the multidrug binding abilities of two out of five gates of the channels deputed to peripheral (early) recognition of substrates. Overall, our findings help to define a common "recognition topology" characterizing Mex transporters, which can be exploited to optimize transport and inhibition propensities of antimicrobial compounds.

A Screening of Antimalarials Extends the Range of Known Escherichia coli AcrB Efflux Substrates and Reveals Two Candidates with Antimicrobial Drug-Enhancing Activity.

Efflux by resistance nodulation cell division transporters, such as AcrAB-TolC in Escherichia coli, substantially contributes to the development of Gram-negative multidrug resistance. Therefore, the finding of compounds that counteract efflux is an urgent goal in the fight against infectious diseases. Previously, an efflux inhibitory activity of the antimalarials mefloquine and artesunate was reported. In this study, we have investigated further antimalarials regarding efflux by AcrB, the pumping part of AcrAB-TolC, and their drug-enhancing potency in E. coli. We show that 10 of the 24 drugs tested are substrates of the multidrug efflux pump AcrB. Among them, tafenoquine and proguanil, when used at subinhibitory concentrations, caused an at least 4- and up to 24-fold enhancement in susceptibility to 6 and 14 antimicrobial agents, respectively. Both antimalarials are able to increase the intracellular accumulation of Hoechst 33342, with proguanil showing similar effectiveness as the efflux inhibitor 1-(1-naphthylmethyl)piperazine. In the case of proguanil, AcrB-dependent efflux inhibition could also be demonstrated in a real-time efflux assay. In addition to presenting new AcrB substrates, our study reveals two previously unknown efflux inhibitors among antimalarials. Particularly proguanil appears as a promising candidate and its chemical scaffold might be further optimized for repurposing as antimicrobial drug enhancer.

Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.

ABSTRACTBacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters.IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.

Diversity and novel mutations in membrane transporters of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), encodes a family of membrane proteins belonging to Resistance-Nodulation-Cell Division (RND) permeases also called multidrug resistance pumps. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in exporting of lipid components across the cell envelope. These proteins perform an essential role in MTB survival; however, there are no data regarding mutations in MmpL, polyketide synthase (PKS) and acyl-CoA dehydrogenase FadE proteins from Khyber Pakhtunkhwa, Pakistan. This study aimed to screen mutations in transmembrane transporter proteins including MmpL, PKS and Fad through whole-genome sequencing (WGS) in local isolates of Khyber Pakhtunkhwa province, Pakistan. Fourteen samples were collected from TB patients and drug susceptibility testing was performed. However, only three samples were completely sequenced. Moreover, 209 whole-genome sequences of the same geography were also retrieved from NCBI GenBank to analyze the diversity of mutations in MmpL, PKS and Fad proteins. Among the 212 WGS (Accession ID: PRJNA629298, PRJNA629388, and ERR2510337-ERR2510345, ERR2510546-ERR2510645), numerous mutations in Fad (n = 756), PKS (n = 479), and MmpL (n = 306) have been detected. Some novel mutations were also detected in MmpL, PKS and acyl-CoA dehydrogenase Fad. Novel mutations including Asn576Ser in MmpL8, Val943Gly in MmpL9 and Asn145Asp have been detected in MmpL3. The presence of a large number of mutations in the MTB membrane may have functional consequences on proteins. However, further experimental studies are needed to elucidate the variants' effect on MmpL, PKS and FadE functions.

Pseudomonas cannabina pv. alisalensis Virulence Factors Are Involved in Resistance to Plant-Derived Antimicrobials during Infection.

Bacteria are exposed to and tolerate diverse and potentially toxic compounds in the natural environment. While efflux transporters are generally thought to involve bacterial antibiotic resistance in vitro, their contributions to plant bacterial virulence have so far been poorly understood. Pseudomonas cannabina pv. alisalensis (Pcal) is a causal agent of bacterial blight of Brassicaceae. We here demonstrated that NU19, which is mutated in the resistance-nodulation-cell division (RND) transporter encoded gene, showed reduced virulence on cabbage compared to WT, indicating that the RND transporter contributes to Pcal virulence on cabbage. We also demonstrated that brassinin biosynthesis was induced after Pcal infection. Additionally, the RND transporter was involved in resistance to plant-derived antimicrobials and antibiotics, including the cabbage phytoalexin brassinin. These results suggest that the RND transporter extrudes plant-derived antimicrobials and contributes to Pcal virulence. We also found that the RND transporter contributes to Pcal virulence on Brassicaceae and tomato, but not on oat. These results suggest that the RND transporter contributes to Pcal virulence differentially depending on the host-plant species. Lastly, our expression-profile analysis indicated that the type-three secretion system (TTSS), which is essential for pathogenesis, is also involved in suppressing brassinin biosynthesis. Taken together, our results suggest that several Pcal virulence factors are involved in resistance to plant-derived antimicrobials and bacterial survival during infection.

A Microbe-Derived Efflux Pump Inhibitor of the Resistance-Nodulation-Cell Division Protein Restores Antibiotic Susceptibility in Escherichia coli and Pseudomonas aeruginosa

The use of efflux pump inhibitors (EPIs) as potentiators along with the traditional antibiotics assists in the warfare against antibiotic-resistant superbugs. Efflux pumps of the resistance-nodulation-cell division (RND) family play crucial roles in multidrug resistance in Escherichia coli and Pseudomonas aeruginosa. Despite several efforts, clinically useful inhibitors are not available at present. This study describes ethyl 4-bromopyrrole-2-carboxylate (RP1) isolation, an inhibitor of RND transporters from the library of 4000 microbial exudates. RP1 acts synergistically with antibiotics by reducing their minimum inhibitory concentration in strains overexpressing archetype RND transporters (AcrAB-TolC and MexAB-OprM). It also improves the accumulation of Hoechst 33342 and inhibits its efflux (a hallmark of EPI functionality). The antibiotic-RP1 combinations prolong the postantibiotic effects and reduce the mutation prevention concentration of antibiotics. Additionally, from Biolayer Interferometry spectra, it appears that RP1 is bound to AcrB. RP1 displays low mammalian cytotoxicity, no Ca2+ channel inhibitory effects, and reduces the intracellular invasion of E.coli and P.aeruginosa in macrophages. Furthermore, the RP1-levofloxacin combination is nontoxic, well-tolerated, and notably effective in a murine lung infection model. In sum, RP1 is a potent EPI and worthy of further consideration as a potentiator to improve the effectiveness of existing antibiotics.

Molecular Determinants of the Promiscuity of MexB and MexY Multidrug Transporters of Pseudomonas aeruginosa.

Secondary multidrug transporters of the resistance-nodulation-cell division (RND) superfamily contribute crucially to antibiotic resistance in Gram-negative bacteria. Compared to the most studied transporter AcrB of Escherichia coli, little is known about the molecular determinants of distinct polyspecificities of the most important RND transporters MexB and MexY of Pseudomonas aeruginosa. In an effort to add knowledge on this topic, we performed an exhaustive atomic-level comparison of the main putative recognition sites (access and deep binding pockets) in these two Mex transporters. We identified an underlying link between some structural, chemical and dynamical features of the binding pockets and the physicochemical nature of the corresponding substrates recognized by either one or both pumps. In particular, mosaic-like lipophilic and electrostatic surfaces of the binding pockets provide for both proteins several multifunctional sites for diffuse binding of diverse substrates. Specific lipophilicity signatures of the weakly conserved deep pocket suggest a key role of this site as a selectivity filter as in Acr transporters. Finally, the different dynamics of the bottom-loop in MexB and MexY support its possible role in binding of large substrates. Our work represents the first comparative study of the major RND transporters in P. aeruginosa and also the first structure-based study of MexY, for which no experimental structure is available yet.

High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

Molecular Rationale behind the Differential Substrate Specificity of Bacterial RND Multi-Drug Transporters

Resistance-Nodulation-cell Division (RND) transporters AcrB and AcrD of Escherichia coli expel a wide range of substrates out of the cell in conjunction with AcrA and TolC, contributing to the onset of bacterial multidrug resistance. Despite sharing an overall sequence identity of ~66% (similarity ~80%), these RND transporters feature distinct substrate specificity patterns whose underlying basis remains elusive. We performed exhaustive comparative analyses of the putative substrate binding pockets considering crystal structures, homology models and conformations extracted from multi-copy μs-long molecular dynamics simulations of both AcrB and AcrD. The impact of physicochemical and topographical properties (volume, shape, lipophilicity, electrostatic potential, hydration and distribution of multi-functional sites) within the pockets on their substrate specificities was quantitatively assessed. Differences in the lipophilic and electrostatic potentials among the pockets were identified. In particular, the deep pocket of AcrB showed the largest lipophilicity convincingly pointing out its possible role as a lipophilicity-based selectivity filter. Furthermore, we identified dynamic features (not inferable from sequence analysis or static structures) such as different flexibilities of specific protein loops that could potentially influence the substrate recognition and transport profile. Our findings can be valuable for drawing structure (dynamics)-activity relationship to be employed in drug design.

The diverse family of MmpL transporters in mycobacteria: from regulation to antimicrobial developments.

Mycobacterial genomes contain large sets of loci encoding membrane proteins that belong to a family of multidrug resistance pumps designated Resistance-Nodulation-Cell Division (RND) permeases. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in the export of lipid components across the cell envelope. These surface-exposed lipids with unusual structures play key roles in the physiology of mycobacteria and/or can act as virulence factors and immunomodulators. Defining the substrate specificity of MmpLs and their mechanisms of regulation helps understanding how mycobacteria elaborate their complex cell wall. This review describes the diversity of MmpL proteins in mycobacteria, emphasising their high abundance in a few opportunistic rapid-growing mycobacteria. It reports the conservation of mmpL loci between Mycobacterium tuberculosis and non-tuberculous mycobacteria, useful in predicting the role of MmpLs with unknown functions. Paradoxically, whereas MmpLs participate in drug resistance mechanisms, they represent also attractive pharmacological targets, opening the way for exciting translational applications. The most recent advances regarding structural/functional information are also provided to explain the molecular basis underlying the proton-motive force driven lipid transport. Overall, this review emphasises the Janus-face nature of MmpLs at the crossroads between antibiotic resistance mechanisms and exquisite vulnerability to drugs.

Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.

Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site.

Mechanistic insight into mycobacterial MmpL protein function.

Mycobacterial cell walls are complex structures containing a broad range of unusual lipids, glycolipids and other polymers, some of which act as immunomodulators or virulence determinants. Better understanding of the enzymes involved in export processes would enlighten cell wall biogenesis. Bernut et al. () present the findings of a structural and functional investigation of one of the most important transporter families, the MmpL proteins, members of the resistance-nodulation-cell division (RND) superfamily. A Tyr842His missense mutation in the mmpL4a gene was shown to be responsible for the smooth-to-rough morphotype change of the near untreatable opportunistic pathogen Mycobacterium bolletii due to its failure to export a glycopeptidolipid (GPL). This mutation was pleiotropic and markedly increased virulence in infection models. Tyr842 is well conserved in all actinobacterial MmpL proteins suggesting that it is functionally important and this was confirmed by several approaches including replacing the corresponding residue in MmpL3 of Mycobacterium tuberculosis. Structural modelling combined with experimental results showed Tyr842 to be a critical residue for mediating the proton motive force required for GPL export. This mechanistic insight applies to all MmpL proteins and probably to all RND transporters.

Heavy metal transport by the CusCFBA efflux system.

It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi-protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu(+) and Ag(+) ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function.

Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections are becoming increasingly difficult to treat due to intrinsic antibiotic resistance and the propensity of this pathogen to accumulate diverse resistance mechanisms. Hyperexpression of efflux pumps of the Resistance-Nodulation-Cell Division (RND)-type multidrug efflux pumps (e.g., MexAB-OprM), chromosomally encoded by mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY (-oprA) is often detected in clinical isolates and contributes to worrying multi-drug resistance phenotypes. Not all antibiotics are affected to the same extent by the aforementioned RND efflux pumps. The impact of efflux on antibiotic activity varies not only between different classes of antibiotics but also between members of the same family of antibiotics. Subtle differences in physicochemical features of compound-pump and compound-solvent interactions largely determine how compounds are affected by efflux activity. The combination of different high-resolution techniques helps to gain insight into the functioning of these molecular machineries. This review discusses substrate recognition patterns based on experimental evidence and computer simulations with a focus on MexB, the pump subunit of the main RND transporter in P. aeruginosa.

Enhancement of antibiotic activity against multidrug-resistant bacteria by the efflux pump inhibitor 3,4-dibromopyrrole-2,5-dione isolated from a Pseudoalteromonas sp.

Members of the resistance nodulation cell division (RND) of efflux pumps play essential roles in multidrug resistance (MDR) in Gram-negative bacteria. Here, we describe the search for new small molecules from marine microbial extracts to block efflux and thus restore antibiotic susceptibility in MDR bacterial strains. We report the isolation of 3,4-dibromopyrrole-2,5-dione (1), an inhibitor of RND transporters, from Enterobacteriaceae and Pseudomonas aeruginosa, from the marine bacterium Pseudoalteromonas piscicida. 3,4-Dibromopyrrole-2,5-dione decreased the minimum inhibitory concentrations (MICs) of two fluoroquinolones, an aminoglycoside, a macrolide, a beta-lactam, tetracycline, and chloramphenicol between 2- and 16-fold in strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM). 3,4-Dibromopyrrole-2,5-dione also increased the intracellular accumulation of Hoechst 33342 in wild-type but not in transporter-deficient strains and prevented H33342 efflux (IC50 = 0.79 μg/mL or 3 μM), a hallmark of efflux pump inhibitor (EPI) functionality. A metabolomic survey of 36 Pseudoalteromonas isolates mapped the presence of primarily brominated metabolites only within the P. piscicida phylogenetic clade, where a majority of antibiotic activity was also observed, suggesting a link between halogenation and enhanced secondary metabolite biosynthetic potential. In sum, 3,4-dibromopyrrole-2,5-dione is a potent EPI and deserves further attention as an adjuvant to enhance the effectiveness of existing antibiotics.

AcrB, AcrD, and MdtABC multidrug efflux systems are involved in enterobactin export in Escherichia coli.

Escherichia coli produces the iron-chelating compound enterobactin to enable growth under iron-limiting conditions. After biosynthesis, enterobactin is released from the cell. However, the enterobactin export system is not fully understood. Previous studies have suggested that the outer membrane channel TolC is involved in enterobactin export. There are several multidrug efflux transporters belonging to resistance-nodulation-cell division (RND) family that require interaction with TolC to function. Therefore, several RND transporters may be responsible for enterobactin export. In this study, we investigated whether RND transporters are involved in enterobactin export using deletion mutants of multidrug transporters in E. coli. Single deletions of acrB, acrD, mdtABC, acrEF, or mdtEF did not affect the ability of E. coli to excrete enterobactin, whereas deletion of tolC did affect enterobactin export. We found that multiple deletion of acrB, acrD, and mdtABC resulted in a significant decrease in enterobactin export and that plasmids carrying the acrAB, acrD, or mdtABC genes restored the decrease in enterobactin export exhibited by the ΔacrB acrD mdtABC mutant. These results indicate that AcrB, AcrD, and MdtABC are required for the secretion of enterobactin.

Bacterial Multidrug Efflux Transporters

Infections caused by bacteria are a leading cause of death worldwide. Although antibiotics remain a key clinical therapy, their effectiveness has been severely compromised by the development of drug resistance in bacterial pathogens. Multidrug efflux transporters--a common and powerful resistance mechanism--are capable of extruding a number of structurally unrelated antimicrobials from the bacterial cell, including antibiotics and toxic heavy metal ions, facilitating their survival in noxious environments. Transporters of the resistance-nodulation-cell division (RND) superfamily typically assemble as tripartite efflux complexes spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusCFBA complex, which mediates resistance to copper(I) and silver(I) ions, is the only known RND transporter specific to heavy metals. Here, we describe the current knowledge of individual pump components of the Cus system, a paradigm for efflux machinery, and speculate on how RND pumps assemble to fight diverse antimicrobials.

Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora.

BackgroundThe Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump.ResultsTo find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment.ConclusionsThe expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in resistance to plant antimicrobials, maybe including flavonoids, which were identified as substrates of both pumps. Furthermore, we found that the mdtABC operon belongs to the regulon of the two-component regulator BaeR suggesting a role of this RND transporter in the cell envelope stress response of E. amylovora.

The Outer Membrane TolC-like Channel HgdD Is Part of Tripartite Resistance-Nodulation-Cell Division (RND) Efflux Systems Conferring Multiple-drug Resistance in the Cyanobacterium Anabaena sp. PCC7120

The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.