Phosphorylcholine stimulates capsule formation of phosphate-limited mucoid Pseudomonas aeruginosa

Abstract

Production of both alginic acid and lipopolysaccharide by a mucoid strain of Pseudomonas aeruginosa, SRM-3, was studied in a chemostat system during growth under nutrient-limiting conditions chosen to reflect the chronic growth conditions in the lungs of cystic fibrosis patients. Since mucoid strains have been shown to elaborate extracellular proteases and phospholipase C, nitrogen and phosphate limitation were selected for analysis. A modified alginate-promoting medium containing either 1 mM glutamate or 0.05 mM K2HPO4 as limiting nutrient and doubling times of 1.6 to 15.7 h were used. Under nitrogen limitation, strain SRM-3 produced 1.4 mg of uronic acid per mg (dry weight) of cells at all doubling times studied. However, phosphate limitation resulted in the synthesis of only 0.4 mg of uronic acid per mg (dry weight) of cells. The role of phosphate in alginic acid polysaccharide production was further investigated by using phosphorylcholine, a product of phospholipase C activity on phosphatidylcholine, the major lung surfactant. Not only were mucoid cells capable of utilizing phosphorylcholine for growth, but a highly specific interaction occurred among phosphorylcholine, alginate, and whole cells, resulting in greatly enhanced culture viscosity. Electron micrographs showed the gradual formation of a capsule during growth on phosphorylcholine, indicating that the mucoid strain has the ability to utilize surfactant not only as a nutrient source but also for constructing a capsule with greatly enhanced adhesive properties.

Environmental conditions which influence mucoid conversion Pseudomonas aeruginosa PAO1

“…Interestingly, 3% of the succinate-grown organisms were with a growth-limiting amount of substrate likely to be found in the lungs was investigated. PC, used previously in phosphate limitation studies with mucoid organisms (18), was used as the growth-limiting phosphate, nitrogen, or carbon source. Also, this compound was evaluated as the sole source of carbon, nitrogen, and phosphate.…”

“…PC is liberated from the major lung surfactant phospholipid phosphatidylcholine by the action of P. aerug- (1). Phosphate-limited cultures of mucoid organisms have been shown to utilize this substance for alginate production and as a source of phosphate for growth (18). PC offers P. aeruginosa the phosphate, carbon, and nitrogen needed for growth.…”

“…Culture media. A modification of the chemically defined alginate-promoting (AP) medium of Mian et al (25), which has been described previously (17,18), was the primary growth medium used for the chemostat studies. The medium contained 100 mM monosodium glutamate, 100 mM sodium gluconate, 7.5 mM NaH2PO4, 16.3 mM K2HPO4, and 10 mM MgSO4 7H20.…”

“…The phenotypic expression of the culture was determined at the same time by noting the number of each colony type on MMA plates. Production of alginate by representative mucoid colonies was verified by isolation and purification of alginate by previously described methods (18), and the stability of mucoid colonies was determined by a minimum of three passages on MMA.…”

“…A chemostat model for the study of this organism under chronic diseaselike growth conditions (phosphate and nitrogen limitation) which would reflect the likely physiological status of organisms in the lungs of CF patients has recently been reported (18). In this study, phosphate-limited cultures of mucoid P. aeruginosa were capable of utilizing phosphorylcholine (PC), a product of phospholipase C activity on phosphatidylcholine (a major component of lung surfactant), as a sole source of phosphate for growth (18).…”