Cyanogenesis in the cystic fibrosis pathogen Pseudomonas aeruginosa

Abstract

Lung disease is the most lethal manifestation of Cystic Fibrosis (CF). The bacterium Pseudomonas aeruginosa is a major pathogen of the CF lung, where it is able to persist for decades. P. aeruginosa is also one of only a few bacterial species capable of producing hydrogen cyanide (HCN). Since cyanide is detectable in the sputum of CF patients, and its presence is negatively correlated with lung function, it has been hypothesised that cyanogenesis in P. aeruginosa may increase virulence in humans. Here, the overall aims have been to explore the clinical relevance of cyanide production in the CF airway and to improve current understanding of the molecular microbiology of cyanogenesis. Ciliary beat frequency (CBF) measurements were performed on nasal brushings and sino-nasal airliquid interface (ALI) cultures obtained from healthy volunteers and CF patients. KCN decreased CBF in healthy nasal brushings (n=6) after 60 min (150µM: 47% fall, p<0.0002; 75µM: 32% fall, P<0.0001). Samples from CF patients (n=3) showed similar results (150µM: 55% fall, P=0.0003). CBF inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited CBF in ALI cultures, albeit to a lesser extent. Additionally, CBF measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants implicated cyanide as a key cilio-inhibitory component. In order to uncover novel determinants of cyanogenesis, a high throughput colourimetric cyanide screen was developed. Cyanogenesis values were obtained for 93% of mutants from a comprehensive nonredundant P. aeruginosa transposon library. Additionally, the validity of two severely impaired mutants, PA2196 and PA5339, was confirmed by complementation. Mapping the most severe cyanogenesis outliers onto the P.aeruginosa genome revealed previously unrelated regions of interest. In particular, overrepresentation of genes related to the motile-sessile switch and to the RoxRS system implicated a strong role for these in the regulation of cyanogenesis.