Conessine as a novel inhibitor of multidrug efflux pump systems in Pseudomonas aeruginosa.
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ABSTRACT: Holarrhena antidysenterica has been employed as an ethnobotanical plant for the treatment of dysentery, diarrhoea, fever, and bacterial infections. Biological activities of the principle compound, conessine including anti-diarrhoea and anti-plasmodial effects were documented. Our previous study reported potency of Holarrhena antidysenterica extract and conessine as resistance modifying agents against extensively drug-resistant Acinetobacter baumannii. This study aimed to investigate (i) whether conessine, a steroidal alkaloid compound, could act as a resistance modifying agent against multidrug-resistant Pseudomonas aeruginosa, and (ii) whether MexAB-OprM efflux pump involved in the mechanism.Conessine combined with various antibiotics were determined for synergistic activity against P. aeruginosa PAO1 strain K767 (wild-type), K1455 (MexAB-OprM overexpressed), and K1523 (MexB deletion). H33342 accumulation assay was used to evaluate efflux pump inhibition while NPN uptake assay was assessed membrane permeabilization.Conessine significantly reduced MICs of all antibiotics by at least 8-fold in MexAB-OprM overexpressed strain. The levels were comparable to those obtained in wild-type strain for cefotaxime, levofloxacin, and tetracycline. With erythromycin, novobiocin, and rifampicin, MICs were 4- to 8-fold less than MICs of the wild-type strain. Loss of MexAB-OprM due to deletion of mexB affected susceptibility to almost all antibiotics, except novobiocin. Synergistic activities between other antibiotics (except novobiocin) and conessine observed in MexB deletion strain suggested that conessine might inhibit other efflux systems present in P. aeruginosa. Inhibition of H33342 efflux in the tested strains clearly demonstrated that conessine inhibited MexAB-OprM pump. In contrast, the mode of action as a membrane permeabilizer was not observed after treatment with conessine as evidenced by no accumulation of 1-N-phenylnaphthylamine.The results suggested that conessine could be applied as a novel efflux pump inhibitor to restore antibiotic activity by inhibiting efflux pump systems in P. aeruginosa. The findings speculated that conessine may also have a potential to be active against homologous resistance-nodulation-division (RND) family in other Gram-negative pathogens.
Project description:In this study, we examined the in vitro effect of tobramycin-efflux pump inhibitor (TOB-EPI) conjugates in combinations with fluoroquinolones, rifampicin and fosfomycin on the growth of multi-drug resistant (MDR) and extremely-drug resistant (XDR) Pseudomonas aeruginosa. The TOB-EPI conjugates include tobramycin covalently linked to 1-(1-naphthylmethyl)-piperazine (NMP) (1), paroxetine (PAR) (2) and a dibasic peptide analogue of MC-04,124 (DBP) (3). Potent synergism was found for combinations of TOB-NMP (1), TOB-PAR (2) or TOB-DBP (3) with either fluoroquinolones (moxifloxacin, ciprofloxacin), rifampicin or fosfomycin against a panel of multidrug-resistant/extensively drug-resistant (MDR/XDR) P. aeruginosa clinical isolates. In the presence of ≤8 mg/L (6.1⁻7.2 µM) (≤¼ × MICadjuvant) concentration of the three conjugates, the MIC80 of moxifloxacin, ciprofloxacin, rifampicin and fosfomycin were dramatically reduced. Furthermore, the MIC80 of rifampicin (0.25⁻0.5 mg/L) and fosfomycin (8⁻16 mg/L) were reduced below their interpretative susceptibility breakpoints. Our data confirm the ability of TOB-NMP (1), TOB-PAR (2) and TOB-DBP (3) conjugates to strongly synergize with moxifloxacin, ciprofloxacin, rifampicin and fosfomycin against MDR/XDR P. aeruginosa. These synergistic combinations warrant further studies as there is an urgent need to develop new strategies to treat drug-resistant P. aeruginosa infections.
Project description:Bacteria have acquired multiple mechanisms to evade the lethal effects of current therapeutics, hindering treatment of bacterial infections, such as those caused by the pathogen Pseudomonas aeruginosa, which is responsible for nosocomial and cystic fibrosis lung infections. One resistance mechanism involves membrane-embedded multidrug efflux pumps that can effectively extrude an array of substrates, including common antibiotics, dyes, and biocides. Among these is a small multidrug resistance (SMR) efflux protein, consisting of four transmembrane (TM) helices, that functions as an antiparallel dimer. TM helices 1 to 3 (TM1 to TM3) comprise the substrate binding pocket, while TM4 contains a GG7 heptad sequence motif that mediates the SMR TM4-TM4 dimerization. In the present work, we synthesized a series of peptides containing the residues centered on the TM4-TM4 binding interface found in the P. aeruginosa SMR (PAsmr), typified by Ac-Ala-(Sar)3-LLGIGLIIAGVLV-KKK-NH2 (helix-helix interaction residues are underlined). Here, the acetylated N-terminal sarcosine (N-methyl-Gly) tag [Ac-Ala-(Sar)3] promotes membrane penetration, while the C-terminal Lys tag promotes selectivity for the negatively charged bacterial membranes. This peptide was observed to competitively disrupt PAsmr-mediated efflux, as measured by efflux inhibition of the fluorescent dye ethidium bromide, while having no effect on cell membrane integrity. Alternatively, a corresponding peptide in which the TM4 binding motif is scrambled was inactive in this assay. In addition, when Escherichia coli cells expressing PAsmr were combined with sublethal concentrations of several biocides, growth was significantly inhibited when peptide was added, notably, by up to 95% with the disinfectant benzylalkonium chloride. These results demonstrate promise for an efflux pump inhibitor to address the increasing threat of antibiotic-resistant bacteria.
Project description:Two new genes (mexXY) similar to mexAB, mexCD, and mexEF and mediating multidrug resistance were cloned from the chromosome of Pseudomonas aeruginosa. Elevated ethidium extrusion was observed with Escherichia coli cells harboring the plasmid carrying mexXY. This MexXY system confers higher resistance to fluoroquinolones than the MexAB and MexCD systems, and E. coli ToIC or P. aeruginosa OprM is necessary for the function of the MexXY system.
Project description:Anti-pseudomonas aminoglycosides, such as amikacin and tobramycin, are used in the treatment of Pseudomonas aeruginosa infections. However, their use is linked to the development of resistance. During the last decade, the MexXY multidrug efflux system has been comprehensively studied, and numerous reports of laboratory and clinical isolates have been published. This system has been increasingly recognized as one of the primary determinants of aminoglycoside resistance in P. aeruginosa. In P. aeruginosa cystic fibrosis isolates, upregulation of the pump is considered the most common mechanism of aminoglycoside resistance. Non-fermentative Gram-negative pathogens possessing very close MexXY orthologs such as Achromobacter xylosoxidans and various Burkholderia species (e.g., Burkholderia pseudomallei and B. cepacia complexes), but not B. gladioli, are intrinsically resistant to aminoglycosides. Here, we summarize the properties (e.g., discovery, mechanism, gene expression, clinical significance) of the P. aeruginosa MexXY pump and other aminoglycoside efflux pumps such as AcrD of Escherichia coli, AmrAB-OprA of B. pseudomallei, and AdeABC of Acinetobacter baumannii. MexXY inducibility of the PA5471 gene product, which is dependent on ribosome inhibition or oxidative stress, is noteworthy. Moreover, the discovery of the cognate outer membrane component (OprA) of MexXY in the multidrug-resistant clinical isolate PA7, serotype O12 deserves special attention.
Project description:The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.
Project description:BackgroundMultidrug resistance Pseudomonas aeruginosa (MDR-P. aeruginosa) is a worldwide threat for public health. Hyperexpression of efflux pump systems (MexAB-OprM and MexCD-OprJ), which is a well-known mechanisms for MDR emerging, is controlled by regulatory genes, mexR and nfxB, respectively. The aim of this study was to evaluate point mutations in mexR and nfxB genes in MDR- P. aeruginosa isolated from wound infections.Materials and methodsA total of 34 P. aeruginosa cultures obtained from wound infections were analyzed. Among them eight isolates identified as MDR-P. aeruginosa and were subjected to determination of mutations in mexR and nfxB genes.ResultsWe detected eight-point mutations in mexR and 12-point mutations in nfxB. The most common mutations were common G327-A (eight isolates), G384-A (eight isolates), G411-A (eight isolates). Mutations in A371-C and A372-C were the predominant substitution which was seen in nfxB. Amino acid substitutions were also found at position 124 and 126 for NfxB and MexR, respectively.ConclusionsP. aeruginosa isolates with mutation in efflux pump regulatory genes such as mexR and nfxB could be a main factor contributed to antibiotic resistance and must be considered in antibiotic treatment.
Project description:Efflux pumps in Gram-negative bacteria such as Pseudomonas aeruginosa provide intrinsic antimicrobial resistance by facilitating the extrusion of a wide range of antimicrobials. Approaches for combating efflux-mediated multidrug resistance involve, in part, developing indirect antimicrobial agents capable of inhibiting efflux, thus rescuing the activity of antimicrobials previously rendered inactive by efflux. Herein, TXA09155 is presented as a novel efflux pump inhibitor (EPI) formed by conformationally constraining our previously reported EPI TXA01182. TXA09155 demonstrates strong potentiation in combination with multiple antibiotics with efflux liabilities against wild-type and multidrug-resistant (MDR) P. aeruginosa. At 6.25 µg/mL, TXA09155, showed ≥8-fold potentiation of levofloxacin, moxifloxacin, doxycycline, minocycline, cefpirome, chloramphenicol, and cotrimoxazole. Several biophysical and genetic studies rule out membrane disruption and support efflux inhibition as the mechanism of action (MOA) of TXA09155. TXA09155 was determined to lower the frequency of resistance (FoR) to levofloxacin and enhance the killing kinetics of moxifloxacin. Most importantly, TXA09155 outperformed the levofloxacin-potentiation activity of EPIs TXA01182 and MC-04,124 against a CDC/FDA panel of MDR clinical isolates of P. aeruginosa. TXA09155 possesses favorable physiochemical and ADME properties that warrant its optimization and further development.
Project description:The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.
Project description:A ceftolozane-tazobactam- and ceftazime-avibactam-resistant Pseudomonas aeruginosa isolate was recovered after treatment (including azithromycin, meropenem, and ceftolozane-tazobactam) from a patient that had developed ventilator-associated pneumonia after COVID-19 infection. Whole-genome sequencing revealed that the strain, belonging to ST274, had acquired a nonsense mutation leading to truncated carbapenem porin OprD (W277X), a 7-bp deletion (nt213Δ7) in NfxB (negative regulator of the efflux pump MexCD-OprJ), and two missense mutations (Q178R and S133G) located within the first large periplasmic loop of MexD. Through the construction of mexD mutants and complementation assays with wild-type nfxB, it was evidenced that resistance to the novel cephalosporin-β-lactamase inhibitor combinations was caused by the modification of MexD substrate specificity.
Project description:BackgroundThe Pseudomonas aeruginosa MexEF-OprN efflux pump confers resistance to clinically significant antibiotics. Regulation of mexEF-oprN operon expression is multifaceted with the MexT activator being one of the most prominent regulatory proteins.MethodologyWe have exploited the impaired metabolic fitness of a P. aeruginosa mutant strain lacking several efflux pump of the resistance nodulation cell division superfamily and the TolC homolog OpmH, and isolated derivatives (large colony variants) that regained fitness by incubation on nutrient-rich medium in the absence of antibiotics. Although the mexEF-oprN operon is uninducible in this mutant due to a 8-bp mexT insertion present in some P. aeruginosa PAO1 strains, the large colony variants expressed high levels of MexEF-OprN. Unlike large colony variants obtained after plating on antibiotic containing medium which expressed mexEF-oprN in a MexT-dependent fashion as evidenced by clean excision of the 8-bp insertion from mexT, mexEF-oprN expression was MexT-independent in the large colony variants obtained by plating on LB alone since the mexT gene remained inactivated. A search for possible regulators of mexEF-oprN expression using transposon mutagenesis and genomic library expression approaches yielded several candidates but proved inconclusive.SignificanceOur results show that antibiotic and metabolic stress lead to up-regulation of MexEF-OprN expression via different mechanisms and that MexEF-OprN does not only extrude antimicrobials but rather serves other important metabolic functions.