Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium

Abstract

The phenazine pigments pyocyanin and 1-hydroxyphenazine were resolved by high-pressure liquid chromatography from the sputum sol phase from 9 of 13 patients with cystic fibrosis or bronchiectasis colonized by Pseudomonas aeruginosa. The concentrations measured were each sufficient to inhibit ciliary beating in vitro and contributed a significant proportion of sol phase toxicity for respiratory epithelium.Pseudomonas aeruginosa colonizes the lungs of patients with cystic fibrosis (CF) and other forms of severe bronchiectasis. We have recently demonstrated that pyocyanin and 1-hydroxyphenazine, phenazine redox pigments generated by P. aeruginosa, disrupt human ciliary beating in vitro (15). Pyocyanin also inhibits epidermal cell growth (4) and lymphocyte proliferation (7), has antibiotic properties against other microorganisms (9), and influences the acquisition of iron by pseudomonads (3). 1-Hydroxyphenazine inhibits mammalian cell respiration (1).Sputum was collected from 12 patients who were colonized by P. aeruginosa; 8 had bronchiectasis and 4 had CF. Pulmonary secretions, from which P. aeruginosa was cultured, were aspirated directly from the large airways of a CF lung removed at transplantation. Sputum was also collected from five patients who had bronchiectasis and who had never been colonized by P. aeruginosa. Each sputum sample was centrifuged (50,000 x g for 90 min at 4°C), the watery sol phase was retained, and the gel phase was discarded. Each sol phase sample (1 ml) was loaded in 5% acetic acid on a C18 Sep Pak column (Waters Associates, Inc.) and eluted with 40% propan-2-ol in aqueous acetic acid. The eluant was fractionated by high-pressure liquid chromatography (HPLC) at 2 ml/min on a p,Bondapak Cl8 column (30 by 0.8 cm) with a 20-min linear gradient from 0 to 40% propan-2-ol in 5% aqueous acetic acid, with monitoring at 280 and 254 nm in UV light (14). Pyocyanin and 1-hydroxyphenazine were identified by their HPLC-UV profiles (14). All pyocyanin concentrations in each sol sample and 1hydroxyphenazine concentrations in sol samples 1, 2, and 7 were calculated after extraction and full UV analysis; 1hydroxyphenazine concentrations in other sol samples were calculated from the HPLC-UV peak height.Human nasal ciliated epithelium was obtained from the inferior turbinate of normal volunteers (8). The sol phase pH was variable (5.5 to 9.5) and was adjusted to 7.4 to ensure that changes in ciliary beat frequency (CBF) were not pH related. A sol phase sample was added to ciliated epithelium, a sealed microscope cover slip slide preparation was constructed, and controls were suspended in phosphate-buffered saline (PBS). Ten epithelium strips were identified, and their positions were marked. CBF was measured in each * Corresponding author. epithelial strip by a photometric technique at 1-h intervals for 4 h (8, 15), and a mean was calculated. Any change in beating pattern, ciliary stasis (previously beating cilia subsequently observed to be static), or change in epithelium structure was noted. Results are sho...

Unsuspected pyocyanin effect in yeast under anaerobiosis

“…For these experiments, we used concentrations of PYO (500 lmol/ L ≡ 105 lg/mL) that are similar to the concentrations previously tested in S. cerevisiae by other authors (Ran et al 2003 [25-100 lg/mL];Angell et al 2006 [25-250 lg/mL]). These concentrations are higher than the concentration of PYO detected in infected lungs of CF patients (Wilson et al 1988;Hunter et al 2012). Notwithstanding, a relative toxin resistance to 500 lmol/L PYO was observed for several genetic backgrounds.…”

“…This difference reinforces the idea that there are probably many different mechanisms of PYO toxicity in living organisms. Nevertheless, it should be kept in mind that the concentration used in anaerobiosis (100 lmol/L) is of the same order as those observed (~10-130 lmol/L or 25-50 lmol/L) in the lungs of CF patients infected by P. aeruginosa (Wilson et al 1988;Hunter et al 2012).…”

Use of the rotating wall vessel technology to study the effect of shear stress on growth behaviour of Pseudomonas aeruginosa PA01

“…The production of pyocyanin and the expression of the cytoplasmic membrane fusion protein mexI were investigated in our study and no significant differences in the tested conditions could be pointed out ( Table 2). The PQS and pyocyanin have been detected in sputum of P. aeruginosa-infected CF patients (Wilson et al, 1988;Collier et al, 2002;Fothergill et al, 2007). However, PQS biosynthesis may vary in function of the stage of P. aeruginosa infection in the CF lung as isolates from patients older than 36 months produce significantly less PQS than those from younger patients (Guina et al, 2003).…”

Targeting virulence factors as an antimicrobial approach: Pigment inhibitors

“…At that time, diphenylamine (DPA, 6), a fatty acid synthetic inhibitor, was identified to partly inhibit the synthesis of carotenoid pigments in several microorganisms at concentrations that do not affect the growth rate of bacteria. Subsequently, many DPA derivatives and P450 inhibitors were synthesized to investigate the effects of inhibiting STX activity ( Figure 4A), such as SKF525A (7), 2-(2,3-dichloro-6-phenylphenoxy)ethylamine (8), and 2-(2,3-dichloro-6-phenylphenoxy)-N,N-diethylamine (9). 84 However, a series of discoveries about STX biosynthesis pathway blockage started from the isoprenoid biosynthesis studies by E. Oldfield's group after the new millennium.…”