The impaired quorum sensing response of MexAB-OprM efflux pump overexpressing mutants is not due to non-physiological efflux of 3-oxo-C12-HSL
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ABSTRACT: Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analyzed efflux pump. Herein, we performed an in-depth analysis of the QS-mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB-OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3-oxo-C12-HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This ‘apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals.
Project description:Efflux pumps of the resistance-nodulation-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 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. The discovered efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, and diminish resistant mutant development. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. 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 no off-target effects and negligible toxicity are potential antibiotic adjuvants to address life-threatening bacterial infections.
Project description:Pseudomonas aeruginosa is an opportunistic human pathogen, which is ubiquitous in the environment. The environmental versatility of P. aeruginosa is often attributed to a large arsenal of active efflux pumps, low permeability of the outer membrane and extensive regulatory networks. In this study, we analyzed putative functional interplay between polyspecific drug efflux pumps of Resistance-Nodulation-Division (RND) superfamily of proteins and GacSA two-component regulatory system. RND transporters are the major contributors to antibiotic resistance and survival under various environmental stresses, whereas GacSA is a global regulator and governs critical lifestyles during human colonization. We found that the inactivation of either RND pumps or gacS induces broad, partially overlapping responses in lifestyle, virulence and metabolic programs that are more pronounced during exponential than stationary phase. GacSA and RND efflux pumps induced opposite responses in gene expression of P. aeruginosa, whereas the overexpression of GacS was additive with the deletion of efflux pumps in gene expression and under growth conditions typically associated with human infections such as elevated temperature and iron deprivation. We conclude that RND efflux and GacSA networks partially overlap with each other.
Project description:Target (MexB) and efflux based mechanisms decreasing the effectiveness of the efflux pump inhibitor D13-9001 in P. aeruginosa PAO1: uncovering a new role for MexMN-OprM in efflux of β-lactams and a novel regulatory circuit (MmnRS) controlling MexMN expression Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in P. aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified and these fell into two categories; those having alterations in the target MexB (F628L and ΔV177) and those with mutations in PA1438 (L172P substitution) which encoded a putative sensor kinase of unknown function. The alterations in MexB were consistent with reported structural studies of D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and >50-fold upregulation of PA1438 and the neighboring response regulator gene PA1437. We propose that these be renamed as mmnR/mmnS for MexMN Regulator and Sensor. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem / biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds was downregulated, suggesting that this channel is also part of the MmnSR regulon. RNA-seq of cells encoding MmnSL172P revealed among other things an interrelationships between regulation of mexMN and genes involved in heavy metal resistance.
Project description:In this study, we investigated the role of efflux pump genes in linezolid resistance. M. tuberculosis H37Rv cultures were exposed to sub-inhibitory concentrations of linezolid (¼ MIC) for 24 hours, and transcriptomic analysis was performed to identify upregulated genes. Of the 120 genes involved in cell wall processes, 9/120 (7.5%) were efflux pump genes, primarily belonging to the ATP-binding cassette (ABC), major facilitator superfamily (MFS), resistance nodulation division (RND), and small multidrug resistance (SMR) families. qRT-PCR, performed at 1/2, 1/4 and 1/8 MIC of linezolid, confirmed the RNA-seq results, showing that 8/9 (88.88%) of the efflux pump genes were upregulated at 1/8 MIC of linezolid, indicating that this concentration is optimal for studying efflux pump activity. These findings not only identify 1/8 MIC as optimum concentration for efflux pump studies after linezolid exposure, they also highlight the significant role of efflux pumps in linezolid resistance, providing potential targets for further research on efflux pumps in clinical isolates of M. tuberculosis.
Project description:The study is about the role of Bacteroides thetaiotaomicron in the human gut microbiota, specifically its ability to form biofilms in response to bile salts. The study found that bile induces the expression of certain efflux pumps, and inhibiting these pumps impairs biofilm formation. Among the induced pumps, the BipABC pump is crucial for biofilm formation as it is involved in the efflux of magnesium, which affects the biofilm's extracellular matrix and structure. This discovery sheds light on how intestinal chemical cues, like bile salts, regulate biofilm formation in B. thetaiotaomicron, a significant gut symbiont.
Project description:Efflux pumps are a significant challenge for the development of new antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, substrate specificities, and the development of inhibitors. However, the complexities of drug efflux networks have limited such studies. To address these challenges, we report the generation of Efflux KnockOut-35 (EKO-35), a highly susceptible Escherichia coli strain lacking 35 efflux pumps. We demonstrate the utility of this strain by constructing an efflux platform consisting of strains individually expressing genes encoding efflux pumps forming tripartite complexes with the outer membrane channel TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux network is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities and also efflux pump interplay. We believe EKO-35 and the efflux platform will have widespread application for the study of drug efflux.
Project description:Purpose: In this study, we analyzed how P. aeruginosa physiology is adapted to the lack of RND-mediated efflux activities. Methods: In this study, we use PΔ6 cells to analyze how P. aeruginosa changes its physiology in response to the lack of efflux pumps and increased permeability of the cell envelope. We compared the transcriptomes of the exponentially growing and stationary PΔ6 and its parent PAO1 cells and identified the cellular functions stressed by the lack of active efflux. High quality total RNA was further processed by removing 23S and 16S rRNAs using the Illumina Ribo-Zero Plus rRNA Depletion kit. Samples were analyzed in duplicate using Illumina MiSeq. Raw data for each sample was analyzed using CLC Genomics Workbench version 12.0.1 software (QIAGEN Aarhus, Denmark). Results: P. aeruginosa PΔ6 strain lacking six best characterized RND pumps activates a specific adaptation response that involves significant changes in expression of specific subset of genes encoding e.g. several transport systems, quorum sensing or iron acquisition. Conclusion: Our results suggest that all changes we observe serve to protect the cell envelope of efflux-deficient P. aeruginosa.
Project description:In light of the antibiotic crisis, emerging strategies to sensitize bacteria to available antibiotics should be explored. Several studies on the mechanisms of killing suggest that bactericidal antibiotic activity is enforced through the generation of reactive oxygen species (ROS lethality hypothesis). Here, we artificially manipulated the redox homeostasis of the model opportunistic pathogen Pseudomonas aeruginosa using specific enzymes that catalyze either the formation or oxidation of NADH. Increased NADH levels led to the activation of antibiotic efflux pumps and high levels of antibiotic resistance. However, higher NADH levels also resulted in increased intracellular ROS and amplified antibiotic killing. Our results demonstrate that growth inhibition and killing activity are mediated via different mechanisms. Furthermore, the profound changes in bioenergetics produced low virulence phenotypes characterized by reduced inter-bacterial signaling controlled pathogenicity traits. Our results pave the way for a more effective infection resolution and add an anti-virulence strategy to maximize chances to combat devastating P. aeruginosa infections while reducing the overall use of antibiotics.
Project description:The number and overlapping substrate repertoire of multidrug efflux pumps in the E. coli genome suggest a physiological role apart from multidrug resistance. This role was investigated using transcriptomic analyses of cDNAs labeled from E. coli AG102 mRNA (hyper drug resistant, marR1) and its isogenic major efflux pump mutants. Keywords: Mutation Analysis
Project description:Purpose: In this study, we analyzed the contributions of AdeABC and AdeIJK in antibiotic resistance and growth physiology of the two MDR strains, AYE and AB5075. Methods: To further characterize the functional interactions between AdeABC and AdeIJK, we carried out RNAseq analysis of the exponential AB5075 and its single and double efflux knockout cells. High quality total RNA was further processed by removing 23S and 16S rRNAs using the MicrobExpress bacterial mRNA enrichment kit. Samples were analyzed in duplicate using Illumina MiSeq. Raw data for each sample was analyzed using CLC Genomics Workbench version 12.0.1 software (QIAGEN Aarhus, Denmark). Results: Our results suggest that inactivation of AdeIJK elicits broader changes in the abundances of mRNAs and this response is modified in the absence of AdeB. In contrast, inactivation of AdeB leads to a focused cellular response, which is not sensitive to the activity of AdeIJK. We identified additional efflux pumps and transcriptional regulators that contribute to MDR phenotype of clinical A. baumannii isolates. Conclusion: Our results suggest that inactivation of AdeIJK elicits broader changes in the abundances of mRNAs and this response is modified in the absence of AdeB. In contrast, inactivation of AdeB leads to a focused cellular response, which is not sensitive to the activity of AdeIJK.