Two Regulators, PA3898 and PA2100, Modulate the Pseudomonas aeruginosa Multidrug Resistance MexAB-OprM and EmrAB Efflux Pumps and Biofilm Formation

Abstract

It is generally believed that the Pseudomonas aeruginosa biofilm matrix itself acts as a molecular sieve or sink that contributes to significant levels of drug resistance, but it is becoming more apparent that multidrug efflux pumps induced during biofilm growth significantly enhance resistance levels. We present here a novel transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux pumps in P. aeruginosa. A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux pumps and phenazine biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) identified its DNA binding sequences and confirmed that PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2. Coimmunoprecipitation revealed a regulatory partner of PA3898 as PA2100, and both are required for binding to DNA in electrophoretic mobility shift assays. PA3898 and PA2100 were given the names MdrR1 and MdrR2, respectively, as novel repressors of the mexAB-oprM multidrug efflux operon and activators for another multidrug efflux pump, EmrAB. The interaction between MdrR1 and MdrR2 at the promoter regions of their regulons was further characterized via localized surface plasmon resonance and DNA footprinting. These regulators directly repress the mexAB-oprM operon, independent of its well-established MexR regulator. Mutants of mdrR1 and mdrR2 caused increased resistance to multiple antibiotics in P. aeruginosa, validating the significance of these newly discovered regulators.

Biofilms as Promoters of Bacterial Antibiotic Resistance and Tolerance

“…However, since pump expression is regulated by multiple mechanisms, it is difficult to speculate about the precise biological role of MarA within the complex regulatory network. In P. aeruginosa , the production of the efflux pumps MexAB-OprM and EmrAB is differentially regulated by the regulators MdrR1 and MdrR2 [ 162 ]. These regulators activate EmrAB but repress MexAB-OprM.…”

There is no hiding if you Seq: recent breakthroughs in Pseudomonas aeruginosa research revealed by genomic and transcriptomic next-generation sequencing

“…ChIP-Seq has also demonstrated novel regulatory pathways linking biofilm and antibiotic resistance. Recently, the transcriptional regulator pair MdrR1 and MdrR2 (previously PA3898 and PA2100) was shown to both control P. aeruginosa biofilm formation through regulation of phenazine biosynthesis and also to directly interact with the promoter region of the mexAB-oprM efflux pump, repressing its transcriptional expression independently of the MexR regulator (37). Not only does P. aeruginosa biofilm block antibiotics with the extracellular matrix, it also controls the transcriptional expression of other antibiotic resistance factors.…”

“…The current standard practice is to prepare transcriptomic or genomic DNA libraries from a population of cells grown in pure culture, but there is much to be gained from "zooming in" to the level of a single cell, or conversely "zooming out" to study mixed cultures containing more than one species of micro-organism. Bacterial single-cell transcriptomics, for which methods are under active development (71,72) will allow exploration of gene expression patterns associated with micro-level differences in location within an infected tissue to a higher resolution than currently possible, or alternatively to explore phenotypic heterogeneity within a population, in particular aspects of phenotypic heterogeneity that are relevant to chronic infections, such as persister cells (73), response to oxygen gradients within a biofilm (74), heterogeneous transcription factor activity patterns (37) or antibiotic tolerance (75). Genomics or transcriptomics performed on multi-species cultures, for example, mixedspecies biofilms or mixed cultures comprising species that are commonly found together in Pseudomonasdominated infection sites, will be able to inform not only about the gene expression patterns and/or essential genomes of the individual pathogens but also about the polymicrobial interactions involved in this mode of growth and conditional essential genotypes required for proliferation in the presence of other species.…”

Cinnamaldehyde Induces Expression of Efflux Pumps and Multidrug Resistance in Pseudomonas aeruginosa

“…Expression of operon mexAB-oprM regulatory genes after exposure of strain PA14 to 256 or 512 g/ml of CNA Repressors MdrR1 and MdrR2 are both required in strain PAO1 for binding to the promoter region of mexAB-oprM(55). However, only mdrR1 expression could be measured because gene mdrR2 is absent from the genome of PA14.…”