Role of the Enterococcus faecalis GelE protease in determination of cellular chain length, supernatant pheromone levels, and degradation of fibrin and misfolded surface proteins - PubMed (original) (raw)

Role of the Enterococcus faecalis GelE protease in determination of cellular chain length, supernatant pheromone levels, and degradation of fibrin and misfolded surface proteins

Christopher M Waters et al. J Bacteriol. 2003 Jun.

Abstract

Gelatinase (GelE), a secreted Zn-metalloprotease of Enterococcus faecalis, has been implicated as a virulence factor by both epidemiological data and animal model studies. Expression of gelE is induced at a high cell density by the fsr quorum-sensing system. In the present study, GelE was shown to be responsible for the instability of a number of Asc10 (aggregation substance) mutant proteins, implying that GelE functions to clear the bacterial cell surface of misfolded proteins. Disruption of GelE production led to increased cell chain length of E. faecalis, from a typical diplococcus morphology to chains of 5 to 10 cells. This function of GelE was also exhibited when the protein was expressed in Streptococcus pyogenes. GelE-expressing E. faecalis strains were more autolytic, suggesting that GelE affects chain length through activation of an autolysin. GelE was also essential for degradation of polymerized fibrin. GelE expression reduced the titer of cCF10, the peptide pheromone that induces conjugation of pCF10, and pCF10 had increased conjugation into non-GelE-expressing strains. These new functions attributed to GelE suggest that it acts to increase the dissemination of E. faecalis in high-density environments.

PubMed Disclaimer

Figures

FIG. 1.

GelE is required for loss of aggregation of Ω2049 and Ω2421 at 37°C. Aggregation of wild-type Asc10 (7517), the vector control (3535), and two temperature-sensitive aggregation insertion mutants (2049 and 2421) grown at 37°C in four E. faecalis strains expressing all combinations of GelE and SprE was determined by OD. 2049 and 2421 expressed in the GelE− strains TX5128 (GelE− SprE−) and TX5264 (GelE− SprE+) resulted in functional aggregation. However, expression of 2049 and 2421 in the GelE+ strains OG1RF (GelE+ SprE+) and TX5243 (GelE+ SprE−) strain did not aggregate. Protease expression had no effect on the aggregation mediated by wild-type Asc10. Bars, from left to right, 7517, 3535, 2049, and 2421.

FIG. 2.

Expression of Asc10 insertion mutants in TX5128. (A) The insertions that resulted in aggregation defects when expressed in OG1RF are shown on a linear map of the gene encoding Asc10. The boxed insertions are those that maintained an aggregation defect when expressed in TX5128. (B) A Western blot of lysozyme surface extracts of insertions in the putative Asc10 aggregation domain expressed in TX5128 confirms full-length protein expression. (7517, wild-type Asc10). Molecular mass markers are indicated to the left of the blot.

FIG. 3.

(A to D) GelE− E. faecalis strains form small chains. Strains that lack GelE production form chains of 5 to 10 cells not usually seen with OG1RF. After crystal violet staining, the cells were visualized at a magnification of ×80 with a phase contrast microscope. (A) OG1RF(GelE+ SprE+); (B) TX5128(GelE− SprE−); (C) TX5264(GelE− SprE+); (D) TX5243(GelE+ SprE−). (E and F) Induction of GelE from a complementing plasmid (3614) restores normal diplococcus morphology. (E) TX5128(3614); (F) TX5264(3614). (G) The relative cell sizes of the bacterial populations were determined by analyzing the FSC of 50,000 events on a flow cytometer. The clear population shift of TX5264 (dark line) versus OG1RF (filled) is evident. Expression of GelE in TX5264 (light line) from 3614 complements the increased cell size and restores it back to OG1RF levels. (H) The mean FSC of three independent cultures of each strain was measured (error bars indicate standard errors of the means). OG1RF-30, OG1RF grown overnight at 30°C.

FIG. 4.

GelE expression in S. pyogenes shortens bacterial chains. Induction of GelE (3614) in strain 90-226 showed a significant (P = 0.032, homoscedastic Student's t test) reduction in chain length versus the vector control (3535) as measured by flow cytometry. (A) Representative histogram of the shift in FSC. (B) Mean FSC and standard error of the mean (SEM) from three independent cultures.

FIG. 5.

GelE-expressing strains are more autolytic. (A) Autolysis of the single protease mutants compared to that of OG1RF. TX5264 was complemented with GelE expressed from plasmid 3614 (plasmid 3535 is the vector control). (B) Autolysis of the double protease mutant TX5128 compared with that of OG1RF. TX5128 was also complemented with plasmid 3614. The data are plotted at each point as the percentage of the initial OD630. Each point represents the mean for four to six independent cultures, and the error bars depict the standard errors of the means.

FIG. 6.

GelE degrades polymerized fibrin. (A) Complete fibrin degradation by the supernatants of strains that express GelE at 24 h. Cultures were grown overnight in the presence of 25 ng of nisin per ml at 37°C, with the exception of well 7, which was grown at 30°C. The supernatants of these cultures were filtered with a 0.45-μm-pore-size syringe tip filter, and a equal volume was added to the polymerized fibrin (time zero). Wells: 1, OG1RF (GelE+ SprE+); 2, TX5128 (GelE− SprE−); 3, TX5264 (GelE− SprE+); 4, TX5243 (GelE+ SprE−); 5, TX5128(3614); 6, TX5264(3614); 7, OG1RF grown at 30°C. (B) The rate of fibrin degradation was also measured by determining the decrease in OD630 of the wells after addition of culture supernatants over the course of 24 h. A representative time course of fibrin degradation is shown. (C) GelE expressed heterologously in L. lactis NZ9800 degrades fibrin at 24 h.

References

1. Bae, T., B. Kozlowicz, and G. M. Dunny. 2002. Two targets in pCF10 DNA for PrgX binding: their role in production of Qa and prgX mRNA and in regulation of pheromone-inducible conjugation. J. Mol. Biol. 315**:**995-1007. -PubMed
1. Beliveau, C., C. Potvin, J. Trudel, A. Asselin, and G. Bellemare. 1991. Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin. J. Bacteriol. 173**:**5619-5623. -PMC -PubMed
1. Bleiweis, A. S., and L. N. Zimmerman. 1964. Properties of proteinase from Streptococcus faecalis var. liquefaciens. J. Bacteriol. 98**:**653-659. -PMC -PubMed
1. Bryan, E. M., T. Bae, H. Kleerebezem, and G. M. Dunny. 2000. Improved vectors for nisin-controlled expression in gram-positive bacteria. Plasmid 44**:**183-190. -PubMed
1. Buttaro, B. A., M. H. Antiporta, and G. M. Dunny. 2000. Cell-associated pheromone peptide (cCF10) production and pheromone inhibition in Enterococcus faecalis. J. Bacteriol. 182**:**4926-4933. -PMC -PubMed

Role of the Enterococcus faecalis GelE protease in determination of cellular chain length, supernatant pheromone levels, and degradation of fibrin and misfolded surface proteins - PubMed (original) (raw)