Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

Abstract

In Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the ⌬anr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the ⌬anr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections. IMPORTANCEPseudomonas aeruginosa causes acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients. P. aeruginosa uses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS, lasR, are frequently observed in clinical isolates. We demonstrated that the regulon attributed to Anr, an oxygen-sensitive transcription factor, was more highly expressed in lasR mutants. Furthermore, we show that Anr regulates the production of several different secreted factors in lasR mutants. These data demonstrate the importance of Anr in naturally occurring quorum sensing mutants in the context of chronic infections. Pseudomonas aeruginosa, a notorious pulmonary pathogen, is frequently a causative agent of nosocomial pneumonias (1), is commonly isolated from the lungs of chronic obstructive pulmonary disease (COPD) patients experiencing exacerbation (2), and is a problematic colonizer of the lungs of individuals with cystic fibrosis (CF) (3). By age 20, 80% of CF patients harbor P. aeruginosa in their lungs (4), and the presence of P. aeruginosa in the airway is correlated with accelerated lung function decline and poor patient prognosis (5, 6). Evidence suggests that in the context of infections, P. aeruginosa is often found in a biofilm state, which contributes to it...

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung

“…A number of transcripts were significantly higher in the LasR Ϫ strains in this in vitro analysis, and most of these transcripts are Anr regulated (see Table S3, sheets 1 and 2) (32). Increased levels of Anr-regulated transcripts in LasR Ϫ strains is consistent with the finding that the level of Anr regulon is higher in strains with QS deficiencies (25).…”

“…5D). Transcription of the cupA1-5 operon and cgrA-C, which encodes the cupA regulator, is controlled by Anr, an oxygen-responsive regulator (25,32,51).…”

“…P. aeruginosa strains were grown either in liquid LB -Lennox for 8 h at 37°C (FRD1, FRD1⌬algU, CI224_M, CI224_nm, CI227, and CI228 strains) or as colony biofilms on tryptone agar for 24 h at 37°C (PAO1, PA14, DH1133, DH2415, PAO1⌬lasR, PA14⌬lasR, DH1132, DH2417, CFRL8, and CFRL11 strains). Data for the wild-type and ⌬lasR strain pairs were also used in analyses published previously (25). Colony biofilms were inoculated from overnight cultures grown in LB medium at 37°C, and colonies were inoculated as 5-l spots.…”

The role for neutrophil extracellular traps in cystic fibrosis autoimmunity

“…P. aeruginosa PA14 and PAO1 strains were provided by Brent Berwin at the Geisel School of Medicine at Dartmouth. The mechanism of BPI cleavage was investigated using PAO1 ΔlasR and PA14 ΔlasR bacterial strains provided by Deborah Hogan at the Geisel School of Medicine at Dartmouth as well as using the PAO1 ΔlasB strain, a gift of Dao Nguyen laboratory at McGill University, Montréal, Quebec, Canada (44,80). PMNs from healthy human donors (1 × 10 6 cells/ml) were treated with PMA (20 nM), glucose oxidase (2 U/ml), or P. aeruginosa PA14 (10 MOI) for 1 hour at 37°C.…”

Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms

“…C4-HSL represses denitrification via its cognate regulator RhlR (69); on the other hand, the addition of PQS to the growth medium specifically promotes NO accumulation (70), thus suggesting a possible mechanism of endogenous regulation of biofilm dispersal (38). Anr, the oxygen-responsive transcription factor controlling the expression of denitrification genes, also controls QS in biofilms under low-oxygen conditions (1%) (73).…”