化膿性鏈球菌是一種常見的人類病原菌，它會藉由不同的機制逃避宿主的免疫組織系統，進而感染宿主產生壞疽性肌膜炎（necrotizing fasciitis）及鏈球菌毒性休克症候群（streptococcal toxic shock syndrome, STSS）。病原體細菌發展了複雜且多樣化的毒性機制削弱或使宿主的免疫防禦系統喪失能力。IdeS (化膿性鏈球菌免疫球蛋白G分解酶)，是從人類的病原菌（化膿性鏈球菌）分泌至細胞外且對免疫球蛋白G具有特異性的半光胺酸蛋白酶（cysteine protease），它會對人類免疫球蛋白G的絞鏈位置進行催化分解作用；IdeS可以藉此幫助化膿性鏈球菌逃離宿主先天的免疫系統及抑制抗體調控的吞噬作用。為了進一步瞭解IdeS酵素活性以及與免疫球蛋白G的作用機制，我們純化重組蛋白質IdeS 野生株、C94S、H262A及S285A突變株並進行特性分析。經由核磁共振的實驗分析得知表現純化後的IdeS具有正確的結構，在研究的過程中發現IdeS在不同的pH範圍由1至11會形成不同的水解產物，並利用N端蛋白質定序得知其氨基酸切位。雖然IdeS是屬於半光胺酸蛋白酶，但研究中發現它不僅可以被半光胺酸蛋白酶抑制劑（IAA, E64）所抑制，還可以被絲胺酸蛋白酶(serine protease)抑制劑（PMSF, TLCK）所抑制。根據氨基酸序列比對，IdeS具有和醣基作用的hemagglutinin domain，因此我們推測免疫球蛋白G上的CH2 domain醣基在IdeS催化過程中扮演重要角色，結果顯示IdeS對不同形式的IgG的催化速率會有差異。利用PNGase F將IgG上醣化去除後IdeS對免疫球蛋白G的活性降低了2倍，而利用DTT將IgG兩個重鏈之間的雙硫鍵去除後IdeS對免疫球蛋白G的活性卻是增高1.5倍，醣基及雙硫鍵一併去除則降低1.7倍。另外我們也發現IdeS及不具蛋白酶活性的C94S皆可以進入人類呼吸道上皮細胞(A549 cell)，並造成細胞凋亡。實驗中也發現caspase抑制劑可以抑制IdeS造成的細胞凋亡，因此IdeS可能會藉由引發caspase cascade進而驅動人類呼吸道上皮細胞產生細胞凋亡的現象，我們的結果顯示IdeS會藉由調整人類的免疫反應及造成細胞凋亡以便增加化膿性鏈球菌在宿主中的存活率上扮演著新的重要機制。

　Streptococcus pyogenes, one of the most common human bacterial pathogens, causes infections such as necrotizing faciitis and streptococcal toxic shock syndrome, by diverse mechanisms that allow the bacteria to evade the immune system and cause disease. Pathogenic bacteria often develop complex and diverse virulence mechanisms that weaken or disable the host immune defense system. The IgG-degrading enzyme of Streptococcus pyogenes (IdeS) is a virulence factor that helps S. pyogenes escaping from human innate immunity and inhibits antibody-mediated phagocytosis. The IdeS, also known as streptococcal Mac, is a secreted IgG-specific cysteine endopeptidase and catalyzes proteolytic cleavage at the lower hinge of human IgG. In order to study the molecular mechanism of IdeS, wild-type, C94S, H262A, and S285A mutant proteins were expressed in E. coli and purified to homogeneity. We found that recombinant IdeS has the correct fold using NMR experiment. Based on the analysis of pH-dependent study, IdeS can be converted into different proteolytic products at pH values ranging from 1 to 11. Three proteolytic products were identified, and their cleavage sites were determined by an N-terminal amino acid sequencer. However, these proteolytic products have similar protease activity. The protease activity of IdeS was inhibited by cysteine protease inhibitors, such as iodoacetate (IAA) and E64. Interestingly, the serine protease inhibitors including PMSF and TLCK can also inhibit its protease activity. The analysis of primary sequence suggested that IdeS contains a hemagglutinin domain involved in glycan binding. Therefore, we hypothesized that two glycosylation sites on IgG CH2 domains plays an important role in recognizing IdeS. Our results showed that IdeS exhibited proteolytic activity to different forms of IgG. For example, deglycosylation of IgG caused a 2-fold decrease in cleavage rate of IgG by IdeS. The removal of disulfide bonds of IgG caused a 1.5-fold increase in cleavage rate of IgG by IdeS. In contrast, the removal of disulfide bonds of deglycosylated IgG caused a 1.7-fold decrease in cleavage rate of IgG by IdeS. We also found that both IdeS and its inactive mutant, C94S, can enter human respiratory epithelial cell A549 and induced cells to undergo apoptosis. Because caspase inhibitors can inhibit IdeS-induced apoptosis in A549 cells, the mechanism may be mediated by activation of caspase cascade. Our results suggest that IdeS represents a novel and significant bacterial virulence determinant that modulates the immune response and induce cell apoptosis to survive in the human host.

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