Effect of ciliostatic factors from Pseudomonas aeruginosa on rabbit respiratory cilia

Abstract

Heat-stable factors released by Pseudomonas aeruginosa in culture supernatants inhibit functional cilia of rabbit tracheal epithelium. Chloroform extraction removed heat-stable factors from stationary-phase culture supernatants. The extracts contained at least seven components separable by thin-layer chromatography (TLC). Cilioinhibitory components were identified as a phenazine derivative, pyo compounds (2-alkyl-4hydroxyquinolines), and a rhamnolipid, also known as a hemolysin. Fluorescence and absorption spectra, relative migration on TLC, staining characteristics, and gas chromatography were the basis for identification. Inhibitory concentrations of each active component were established by quantitative measures of percent motility and beat frequency. Corresponding damage to ciliary ultrastructure was examined by electron microscopy. The pyo compounds produced ciliostasis at concentrations of 50 ,ug/ml, but without obvious ultrastructural lesions. The phenazine derivative also inhibited ciliary motility and caused some membrane disruption, although at substantially greater concentrations of 400 ,ig/ml. Limited exposure of tracheal explants to the rhamnolipid resulted in ciliostasis which was associated with altered ciliary membranes. More extensive exposure to rhamnolipid was associated with removal of dynein arms from axonemes. Pyocyanin at a concentration of 0.5 mg/ml did not inhibit ciliary beating under our conditions. The data suggest that the pyo compounds are the most effective per weight ciliostatic factors released by P. aeruginosa and rhamnolipid is the most destructive of cilia ultrastructure. By interfering with normal ciliary function, these ciliostatic factors may enable P. aeruginosa to more easily colonize the respiratory tract. * Corresponding author.Ultrastructural observations were correlated with the effect on ciliary activity. MATERIALS AND METHODSCilia motility assay. Rabbit tracheal explants were used to quantitate ciliostatic activity. New Zealand White male rabbits were killed by intravenous injection of 150 mg of pentobarbital sodium (Nembutal; Abbott Laboratories, North Chicago, Ill.), the neck and thorax were dissected, and the trachea was removed. After an initial washing in saline, the trachea was cut first along the membranous portion and then between the cartilage, forming open rings approximately 1.0 mm in thickness. The cut tracheal rings were incubated in minimal essential medium with Earle salts (GIBCO Laboratories, Grand Island, N.Y.) containing streptomycin (0.13 mg/ml), penicillin G (0.063 mg/ml), and mycostatin (50 U/ml) at 37°C in 5% CO2 until use. The tracheal rings were rinsed with saline and placed in 96-well flat-bottom microtiter plates containing 0.2 ml of Hanks balanced salt solution (Flow Laboratories, Inc., McLean, Va.) or a buffered salt solution (0.02 M Tris hydrochloride, 0.15 M KCl, 2.0 mM MgSO4, 5 mM dithiothreitol [pH 7.6]) at room temperature. Identical results were obtained with either buffer. With an inverted microscope (x 200, Olympus IM; New Jersey...

Interference in adhesion of bacteria and yeasts isolated from explanted voice prostheses to silicone rubber by rhamnolipid biosurfactants

“…Low concentrations of purified P. aeruginosa rhamnolipids were found to induce changes in fluid transport across airway surface epithelia, thus encouraging colonization by increasing the number of binding sites for P. aeruginosa (Graham et al 1993, Evans et al 1998. Moreover, rhamnolipids were found to be ciliostatic factors that may enable P. aeruginosa to more easily colonize the respiratory tract predisposing the lung to infection (Hingley et al 1986, Read et al 1992. Therefore, further work has to be done in order to guarantee that the studied rhamnolipid will not produce undesirable effects in the oropharyngeal environment.…”

Pseudomonas aeruginosarhamnolipids disperseBordetella bronchisepticabiofilms

“…Based on a modified protocol developed from previous reports [22,23], we purified rhamnolipids from the culture medium of wild type P. aeruginosa PAO1 and also made a control preparation from the rhlA mutant. The rhamnolipid preparations were examined with thin-layer chromatography based on a published protocol [31], and we observed the expected rhamnolipid staining on the TLC plate only from the preparation of the wild type bacterial culture (data not shown).…”

Mimicking the host and its microenvironmentin vitrofor studying mucosal infections byPseudomonas aeruginosa

“…These findings highlight the importance of mimicking mucociliary clearance in vitro . Most publications describing the effect of P. aeruginosa and its virulence factors (such as lectins, rhamnolipids, proteases, and phenazines) on ciliary function have used primary respiratory epithelium grown as ALI or IVOCs (Wilson et al ., ; Hingley et al ., , b; Munro et al ., ; Read et al ., ; Bajolet‐Laudinat et al ., ). Indeed, these models contain ciliated cells at ratios similar to the respiratory mucosa.…”