Abstract
In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. In addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, we compared the transcriptional profile generated using DNA microarrays of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought, with the deletion of ampR influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Other virulence mechanisms including biofilm formation and QS-regulated acute virulence factors are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the microarray data. Further, using a Caenorhabditis elegans model, we demonstrate that a functional AmpR is required for P. aeruginosa pathogenicity. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. Further, we show differential regulation of other transcriptional regulators and sigma factors by AmpR, accounting for the extensive AmpR regulon. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating biofilm formation, a chronic infection phenotype. Unraveling this complex regulatory circuit will provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors in response to antibiotic exposure.
Highlights
Pseudomonas aeruginosa is one of the leading opportunistic Gramnegative nosocomial pathogens
Personal communication). bLactamase quantification showed that PAODampR produced significantly lower amounts in response to b-lactam stress compared to PAO1 (PAO1: 11.27 mU vs. PAODampR: 6.5 mU, p value 0.0003; Fig. 1B), which is in agreement with the E-test data
The results demonstrate that AmpR influences the expression of 313 genes in the absence of b-lactam stress and an additional 207 genes when exposed to sub-minimum inhibitory concentration (MIC) b-lactam stress
Summary
Pseudomonas aeruginosa is one of the leading opportunistic Gramnegative nosocomial pathogens. This is true in critically ill patients, where multi-drug resistant P. aeruginosa is a severe problem. It is the leading pathogen in ventilator-associated pneumonia with a mortality rate of 40–60% [1]. Inability to eradicate the infection is partly due to intrinsic and acquired antibiotic resistance of P. aeruginosa. Antibiotic resistant isolates of P. aeruginosa are selectively favored in vivo in CF patients [8,9]. Resistance of P. aeruginosa to the b-lactam class of antibiotics, currently used to treat P. aeruginosa infections, is partly mediated by a group of genes belonging to the amp system
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