RND-type Pumps Research Articles

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.

Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time.

The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves—causing an increased drug efflux efficiency—are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors.

Emergence of a Plasmid-Encoded Resistance-Nodulation-Division Efflux Pump Conferring Resistance to Multiple Drugs, Including Tigecycline, in Klebsiella pneumoniae.

Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K.pneumoniae, Escherichia coli, and Salmonella TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniaetmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning.IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings-especially carbapenem-resistant K.pneumoniae Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a "One Health" perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

Phylogenetic and functional characterisation of the Haemophilus influenzae multidrug efflux pump AcrB

Multidrug resistance in Gram-negative bacteria can arise by the over-expression of multidrug efflux pumps, which can extrude a wide range of antibiotics. Here we describe the ancestral Haemophilus influenzae efflux pump AcrB (AcrB-Hi). We performed a phylogenetic analysis of hundreds of RND-type transporters. We found that AcrB-Hi is a relatively ancient efflux pump, which nonetheless can export the same range of antibiotics as its evolved colleague from Escherichia coli. AcrB-Hi was not inhibited by the efflux pump inhibitor ABI-PP, and could export bile salts weakly. This points to an environmental adaptation of RND transporters. We also explain the sensitivity of H. influenzae cells to β-lactams and novobiocin by the outer membrane porin OmpP2. This porin counterbalances the AcrB-Hi efflux by leaking the drugs back into the cells. We hypothesise that multidrug recognition by RND-type pumps is not an evolutionarily acquired ability, and has been present since ancient promiscuous transporters.

Recent advances toward a molecular mechanism of efflux pump inhibition.

Multidrug resistance (MDR) in Gram-negative pathogens, such as the Enterobacteriaceae and Pseudomonas aeruginosa, poses a significant threat to our ability to effectively treat infections caused by these organisms. A major component in the development of the MDR phenotype in Gram-negative bacteria is overexpression of Resistance-Nodulation-Division (RND)-type efflux pumps, which actively pump antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of RND-type efflux pump have been reported in the literature, and at least three of these EPI series were optimized in a pre-clinical development program. However, none of these compounds have been tested in the clinic. One of the major hurdles to the development of EPIs has been the lack of biochemical, computational, and structural methods that could be used to guide rational drug design. Here, we review recent reports that have advanced our understanding of the mechanism of action of several potent EPIs against RND-type pumps.

Functional study of the novel multidrug efflux pump KexD from Klebsiella pneumoniae

We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.

Characterization of the RND-Type Multidrug Efflux Pump MexAB-OprM of the Plant Pathogen Pseudomonas syringae

In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In Pseudomonas aeruginosa, a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens Pseudomonas syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. syringae pv. tomato DC3000 were identified, and mexAB-oprM-deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the mexAB-oprM-deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the mexAB-oprM operon in P. syringae; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.

A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps

Bacteria such as Escherichia coli and Pseudomonas aeruginosa expel antibiotics and other inhibitors via tripartite multidrug efflux pumps spanning the inner and outer membranes and the intervening periplasmic space. A key event in pump assembly is the recruitment of an outer membrane-anchored TolC exit duct by the adaptor protein of a cognate inner membrane translocase, establishing a contiguous transenvelope efflux pore. We describe the underlying interaction of juxtaposed periplasmic exit duct and adaptor coiled-coils in the widespread RND-type pump TolC/AcrAB of E. coli, using in vivo cross-linking to map the extent of intermolecular contacts. Cross-linking of site-specific TolC cysteine variants to wild-type AcrA adaptor identified residues on the lower alpha-helical barrel domain of TolC, defining a contiguous cluster close to the entrance aperture of the exit duct. Reciprocally, site-specific cross-linking of AcrA cysteine variants to wild-type TolC identified the interaction surface on the adaptor within the N-terminal alpha-helix of the AcrA coiled-coil. The experimental data allowed a data-driven docking approach to model the interaction surface central to pump assembly. The lowest energy docked model satisfying all of the cross-link distance constraints places the adaptor at the intramolecular groove formed by the TolC entrance helices, aligning the adaptor coiled-coil with the exposed TolC outer helix. A key feature of this positioning is that it allows space for the proposed movement of the inner coil of TolC during transition to its open state.

Mammalian Steroid Hormones Are Substrates for the Major RND- and MFS-Type Tripartite Multidrug Efflux Pumps of Escherichia coli

A steroid-hormone-dependent growth suppression was observed in Escherichia coli efflux-deficient backgrounds containing mutations in the major RND- and MFS-type tripartite multidrug efflux systems, AcrAB-TolC and EmrAB-TolC, respectively. In addition to their previously known natural steroid spectrum, which includes bile acids, both systems were shown to transport the hormones estradiol and progesterone, whereas hydrocortisone served as a substrate of only AcrAB-TolC. Furthermore, at least two other RND-type pumps, YhiV and AcrD, were capable of transporting such hormones when overexpressed on plasmid vectors (with some demonstrable specificity observed with AcrD). When this activity was examined in a wild-type background, cell-associated estradiol levels remained largely unaffected by competition with exogenous bile acids and hydrocortisone, in contrast to progesterone, which produced a significant modulation in estradiol uptake.