Calcium and magnesium enhance the production of Pseudomonas aeruginosa protease IV, a corneal virulence factor

Abstract

The effect of calcium and magnesium on protease IV production during the growth of Pseudomonas aeruginosa was investigated. Strain PA103 was grown to stationary phase in medium containing various concentrations of either calcium or magnesium. Culture supernatants were concentrated, standardized relative to cell density, and the pyoverdine concentrations were measured. Overall extracellular protease activity and specific protease IV (lysine endoproteinase) activity were measured with or without TLCK, a serine protease inhibitor effective against protease IV activity. Protease IV activity was also observed by casein zymography. Calcium and magnesium were quantified in the corneas and aqueous humor of rabbits that were inoculated intrastromally with strain PA103. Pyoverdine production was not significantly different in cultures grown in medium with added calcium or magnesium, but extracellular caseinase activity increased in these cultures. Susceptibility of caseinase activity to TLCK inhibition and a specific assay for protease IV indicated that protease IV activity increased in cultures grown in calcium or magnesium. Casein zymography supported the observation that protease IV activity increased in the cultures with added calcium and magnesium. Addition of calcium or magnesium to the protease IV-specific assay had no effect on the catalytic activity of pure protease IV. Infection of rabbit corneas with PA103 did not change the magnesium concentration in either corneas or aqueous humor, but significantly increased the concentration of calcium in corneas. These results indicate that calcium and magnesium enhance the production of protease IV, but not pyoverdine production. Calcium increases in the cornea following infection with P. aeruginosa could favor production of protease IV.

Strain-specific proteome responses of Pseudomonas aeruginosa to biofilm-associated growth and to calcium

“…PrpL was also induced by Ca 2+ in planktonic FRD1 cells. Previously PrpL has been shown to be Ca 2+ -regulated in P. aeruginosa PA103 (Marquart et al, 2005). PrpL is regulated by PvdS (Wilderman et al, 2001), which also regulates pyoveridine production.…”

“…It also plays a signalling role in bacteria, particularly in the case of secreted proteins (Marquart et al, 2005;Olson & Ohman, 1992;Sarkisova et al, 2005;Yahr et al, 1997). Since Ca 2+ influences biofilm architecture and production of extracellular matrix materials in P. aeruginosa, we chose to continue our studies on the physiological differences of biofilm-associated bacteria by investigating the effect of Ca 2+ and biofilm growth of the P. aeruginosa cytosolic proteome.…”

Protease-Stable Polycationic Photosensitizer Conjugates between Polyethyleneimine and Chlorin(e6) for Broad-Spectrum Antimicrobial Photoinactivation

“…This means that they are likely to be substrates for protease enzymes that are found in tissue. In particular, extracellular proteases derived from the host cells are found in areas of inflammation such as infections (1,23) and proteases may also be produced by bacteria them-selves as secreted virulence factors involved in tissue invasion (30,47).…”

Identification of a Novel Secreted Protease fromPseudomonas aeruginosathat Causes Corneal Erosions

“…22,29 Its production is augmented with increased calcium or magnesium in the growth medium. 30 Alkaline protease is a metalloprotease of P. aeruginosa. The role of this protease in keratitis has not been fully elucidated.…”