Mucin degradation mechanisms by distinct Pseudomonas aeruginosa isolates in vitro

Abstract

Pseudomonas aeruginosa has emerged as an important causative agent of bacterial keratitis, a rapidly progressive ocular condition that may result in blindness. Secretory mucin forms the main constituent of the precorneal tear film, a three-layer film on the ocular surface protecting the underlying corneal epithelium from potential pathogens. The purpose of the present study was to compare mucin degradation mechanisms between ocular P. aeruginosa strains. Mucin degradation was assessed by agarose electrophoresis, lectin blotting, and size exclusion chromatography. The results indicate that certain P. aeruginosa strains (Paer12, ATCC 15442, 6294, and Paer25) had depleted mucin from the culture supernatant and that this was contingent on the inherent ability of these isolates to produce proteases. Non-protease-producing strains (Paer1 and Paer3) did not appreciably degrade mucin. Further, galactosidase, N-acetylglucosaminidase, and N-acetylgalactosaminidase activities were detected in some strains, suggesting the operation of further mechanisms of mucin degradation by P. aeruginosa. Mucin degradation by P. aeruginosa also seemed to be for the acquisition of nutrients, as a growth advantage was observed in mucin-depleting strains over nondepleting strains in the long term. It is postulated that the degradation of mucin serves to collapse the mucin barrier and its associated network containing antibacterial tear components and to provide energy for sustained bacterial growth.

FIG. 1.

Comparison of mucin degradation between P. aeruginosa isolates at 0 and 24 h. Culture supernatants were loaded onto a 1% agarose gel and stained with biotin-labelled WGA lectin to detect mucin components on the blot. The mucin control included the incubation of PGM alone without bacteria.

FIG. 2.

SDS-PAGE protein profile of culture supernatants after incubation of P. aeruginosa strains with mucin. (A) Reference protein profiles of mucin alone and purified elastase in mucin. (B and C) Culture supernatants of representative strains Paer1 (B) and 6294 (C) in mucin at various time points. Samples were applied to a 4 to 15% polyacrylamide gel under reducing and denaturing conditions and stained with silver. Lanes: 1, 0 h; 2, 2 h; 3, 4 h; 4, 6 h; 5, 8 h; 6, 10 h; 7, 24 h; 8, 48 h; 9, 72 h. The arrow indicates the position of low-molecular-weight material depleted by strains over time. The arrowhead indicates the position of the “link” protein.

FIG. 3.

Gel filtration profile of spent culture supernatant after incubation of strain 6294 in mucin for 24 h (A) and 72 h (B). The control included the incubation of 1% (wt/vol) PGM in PBS without the inoculation of bacteria. ⧫, mucin control (no bacteria); ◊, mucin with strain 6294.

FIG. 4.

Zymograms of protease production by P. aeruginosa isolates after incubation with PGM for 24 h (A) and 72 h (B). Lanes: 1, mucin control; 2, Paer1; 3, Paer3; 4, Paer12; 5, ATCC 15442; 6, 6294; 7, Paer25.

FIG. 5.

Glycosidase production by P. aeruginosa strains after incubation with PGM for 72 h. One unit of enzyme activity is defined as the amount of enzyme catalyzing the release of 1 nmol of pNP, and values were corrected for background levels of the mucin control culture supernatant containing no bacteria.

FIG. 6.

Use of mucin as the sole carbon and nitrogen source for growth of P. aeruginosa isolates. (A) Control incubations of representative strain 6294 relative to growth in mucin. ⧫, 6294 with mucin in PBS; ◊, 6294 in PBS containing ammonium and glucose; ▴, 6294 in PBS alone. (B) Growth of various P. aeruginosa isolates in mucin. Data are presented as mean values from at least two independent experiments. Arrows indicate two strains having no significant increase in growth between 48 and 72 h.