Effect of group A streptococcal cysteine protease on invasion of epithelial cells

Abstract

Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the speB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells.

FIG. 1

Southern blot analysis of genomic DNA extracted from GAS A-20 and SW507. The probes used were specific for the speB gene and λ DNA. The DNAs were digested with NsiI (lanes 1 and 4), HindIII (lanes 2 and 5), and HindIII-NsiI (lanes 3 and 6). Lanes 1 to 3, GAS A-20 genomic DNA; lanes 4 to 6, GAS SW507 genomic DNA; lanes M, λ HindIII marker used as a molecular size standard.

FIG. 2

Western immunoblot analysis of SPE B present in the supernatants of wild-type GAS (A-20 and NZ131) and mutants (SW507 and SW510) after electrophoresis on an 8% polyacrylamide gel. The blot was developed with rabbit anti-SPE B polyclonal antibodies. Lane 1, A-20; lane 2, SW507; lane 3, NZ131; lane 4, SW510; lane 5, purified 28-kDa SPE B from A-20 used as a standard. The mature SPE B protein is indicated (arrow).

FIG. 3

Growth curves for wild-type strains (A-20 and NZ131) and isogenic mutants (SW507 and SW510) in TSBY (A) and DMEM (B).

FIG. 4

Electron micrograph of GAS strain A-20 entry into cultured A-549 cells. Monolayers were infected with 5 × 10⁷ CFU for 120 min before they were washed and exposed to penicillin. A bacterium (B) is enclosed in a vacuole (V). The nucleus (N) and microvilli (MV) are indicated. Magnification, ×10,000.

FIG. 5

Invasion kinetics of the wild-type strains (A-20 and NZ131) and isogenic mutants (SW507 and SW510). Confluent cell monolayers were infected with 5 × 10⁷ bacteria in 24-well plates. The cells were incubated for 1.5, 2, 3, 4, 5, and 10 h, washed with PBS, and incubated for another 1.5 h in medium containing 0.8 μg of penicillin per ml. GAS cell numbers were determined by serial dilutions of cell lysates and plating on agar. Results are means ± standard errors of the means (SEM) of the numbers of GAS per well from four separate experiments; at each time point, triplicate samples were analyzed.

FIG. 6

Intracellular growth of S. pyogenes in A-549 cells. Confluent cell monolayers were infected with 5 × 10⁷ bacteria in 24-well plates. The cells were incubated for 90 min, washed, and incubated for another 1.5 to 4 h in medium containing 0.8 μg of penicillin per ml. GAS cell numbers were determined by serial dilution of cell lysates and plating on agar. Results are the means ± SEM of the numbers of GAS per well from four separate experiments; at each time point, triplicate samples were analyzed.