| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 87 === Streptococcus pyogenes, group A streptococcus (GAS), is the major causative agent of a number of human diseases. Since 1980, striking increase in the frequency and severity of streptococcal infections including soft tissue necrosis, streptococcal toxic shock-like syndrome (STSS), and scarlet fever is worldwide. However, there were only a few reports in the pathogenesis of GAS in Taiwan. To determine whether the spectrum of invasive GAS diseases in Taiwan parallels the increasing incidence and severity seen in other countries, we conducted a 4-year study reviewing all invasive GAS infections at two hospital centers in southern Taiwan. Neither the presence of speA, speC, or speF of the isolates is implicated in any particular clinical syndrome of patients with invasive GAS diseases. High protease activity and M1 serotype of isolates were significantly associated with clinical signs of STSS and mortality. The M1 serotype and protease activity, as well as the host immunity status may play significant roles in the pathogenesis of invasive diseases in Taiwan. Several lines of evidence suggest that streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, plays an important role in GAS infection. In order to understand the role of protease in the pathogenesis of streptococcal infection and the relationship between invasion and protease, I have established an internalization assay on the basis of resistance of intracellular streptococci to penicillin and the internalization was confirmed by the electron microscope. Isogenic protease mutants were constructed by using integrational plasmid to disrupt the speB gene. The mutants had growth rates similar to those of the wild-type strains. When invading A-549 cells, the mutants had a two- to threefold decrease in invasion activity compared to that of the wild-type strains. The invasion 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 adhesive ability of GAS. These results suggest that streptococcal cysteine protease can enhance the invasion of GAS into human respiratory epithelial cells. The fate of the GAS-infected epithelial cells was further studied. GAS has the ability to invade A-549 and HEp-2 cells. Both A-549 and HEp-2 cells were killed by infection with GAS. Epithelial cell death mediated by GAS at least in part through apoptosis, as shown by cellular morphology, staining with propidium iodide, and DNA fragmentaion. Apoptosis can be blocked by using cytochalasin D which interferes with cytoskeleton function. The caspase inhibitors, Z-VAD.CMK, Ac-YVAD.FMK, and Ac-DEVD.FMK, inhibited GAS-induced apoptosis. I further examined whether SPE B is involved in the induction of apoptosis. The speB isogenic mutants had less ability to induce cell death than those of the wild-type strains. When the A-549cells were cocultured with the mutant and SPE B for 2 h, the number of intracellular bacteria increased to the level of wild-type strains, whereas, the percentage of apoptotic cells did not increase. These results demonstrate for the first time that GAS induces apoptosis of epithelial cells and internalization is required for apoptosis. Th caspase pathway is involved in GAS-induced apoptosis, and expression of SPE B within the cells enhances apoptosis. This study provides a novel information regarding GAS interacting with the epithelial cells, and opens a new spectrum to study the pathogenic mechanism of GAS.
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