Pseudomonas aeruginosa regulates flagellin expression as part of a global response to airway fluid from cystic fibrosis patients

Abstract

Cystic fibrosis (CF) patients are highly susceptible to chronic lung infections by the environmental bacterium Pseudomonas aeruginosa. The overproduction and accumulation of dehydrated viscous respiratory mucus and excessive inflammation represents a defining feature of CF and constitutes the major environment encountered by P. aeruginosa during chronic infections. We applied whole-genome microarray technology to investigate the ability of P. aeruginosa to respond to signals found in muco-purulent airway liquids collected from chronically infected CF patients. Particularly notable was the activation of the Rhl-dependent quorum-sensing (QS) network and repression of fliC, which encodes flagellin. Activation of the Rhl branch of the QS network supports the observation that QS molecules are produced in the chronically infected CF lung. The shut-off of flagellin synthesis in response to CF airway liquids was rapid and independent of QS and the known regulatory networks controlling the hierarchical expression of flagellar genes. As flagellin is highly immunogenic and subject to detection by host pattern recognition receptors, its repression may represent an adaptive response that allows P. aeruginosa to avoid detection by host defense mechanisms and phagocytosis during the chronic phase of CF lung infections.

Fig. 1.

Cluster analysis (14) of P. aeruginosa genes showing altered expression after growth in the presence of MPM collected from the airways of chronically infected CF patients. Indicated in the first and second columns are genes showing a >5-fold change in expression in M63 medium containing 10% MPM collected from two different CF patients relative to M63 alone (samples 1 and 2, respectively). Genes represented in green are repressed in response to signal(s) present in the MPM samples. Genes in red are activated. The role of the Rhl and Las QS systems in controlling the expression of these MPM-responsive genes was determined by comparing gene expression in a rhlR, lasR double mutant relative to wild type, both in the presence of 10% MPM (sample 1). In the third column, genes represented in green require the QS system for activation in MPM, genes in red require QS for repression in MPM and genes in black are QS-independent (<2-fold change). Black lines indicate genes previously shown to be regulated by the QS system (15, 16). Black dots denote genes specifically regulated by the Rhl QS system as inferred from Schuster and coworker (15).

Fig. 2.

Exposure to CF derived muco-purulent respiratory material results in transcriptional repression of fliC, encoding flagellin. Electron microscopic observation indicates that polar flagellar filaments, composed of flagellin (arrows) are present (A) on the surface of P. aeruginosa grown in M63 medium and absent (B) from the surface of bacteria grown in the same medium containing 10% MPM. (C) Immunoblot analysis of whole-cell protein samples with flagellin-specific antiserum confirms that P. aeruginosa grown in the presence of 10% MPM produce significantly reduced amounts of flagellin (arrow) compared to equivalent samples derived from bacteria grown in medium lacking MPM. No flagellin is detectable in protein samples from bacteria lacking the flagellin gene fliC. (D) Reverse transcription polymerase chain reactions with fliC-specific oligonucleotide primers and total mRNA extracted from wild-type P. aeruginosa at 0, 2, and 4 h after subculture in M63 medium with or without 10% MPM. Samples were equalized based on RT-PCR products for the constitutively expressed clpX gene.

Fig. 3.

The mechanism of fliC repression in response to MPM likely acts through the alternative sigma factor FliA. (A) Presented is a comparison of the transcriptional response of flagellar biogenesis genes in mutants lacking known flagellar regulators relative to wild type and the expression change in wild type grown in 10% MPM relative to medium lacking MPM. The transcriptional response is given as the average fold change for each of the 14 flagellar biogenesis gene operons (11). Expression data for the flagellar regulatory mutants was derived from previously published transcriptomes (11). Black bars, rpoN mutant vs. wild type; red bars, fleQ mutant vs. wild type; blue bars, fleR mutant vs. wild type; green bars, fliA mutant vs. wild type; yellow bars, wild type grown in M63 containing 10% MPM vs. M63 alone. (B) Response of a fliC::lacZ transcriptional reporter in wild type and flagellar mutants in the absence and presence of 10% MPM.

Fig. 4.

Environment-specific QS gene response. Cluster analysis (14) of genes that show CF respiratory liquid-specific patterns of QS-dependent expression not previously identified under laboratory growth conditions. Indicated in the first column are genes showing a >5-fold change in expression in wild-type P. aeruginosa relative to a rhlR, lasR double mutant, both grown in M63 medium containing 10% MPM. Green and red depict QS-dependent genes that are repressed or activated, respectively, in 10% MPM. The second through fifth columns represent the expression pattern of these genes at the indicated culture densities (OD₆₀₀) in laboratory medium supplemented with synthetic autoinducers (rhlI, lasI mutant with added autoinducers versus no autoinducer). Genes in green and red are repressed and activated, respectively, by the addition of exogenous autoinducers. Genes in black are QS-independent (<2-fold change). Data for growth phase-dependent gene expression in the presence of P. aeruginosa QS autoinducers has been published (15).