Juarez Z E, Stinson M W
Center for Microbial Pathogenesis, School of Medicine and Biomedical Sciences, State University of New York at Buffalo 14214, USA.
Infect Immun. 1999 Jan;67(1):271-8. doi: 10.1128/IAI.67.1.271-278.1999.
Streptococcus gordonii is a frequent cause of infective bacterial endocarditis, but its mechanisms of virulence are not well defined. In this study, streptococcal proteases were recovered from spent chemically defined medium (CDM) and fractionated by ammonium sulfate precipitation and by ion-exchange and gel filtration column chromatography. Three proteases were distinguished by their different solubilities in ammonium sulfate and their specificities for synthetic peptides. One of the enzymes cleaved collagen analogs Gly-Pro 4-methoxy-beta-naphthylamide, 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), and p-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (pZ-peptide) and was released from the streptococci while complexed to peptidoglycan fragments. Treatment of this protease with mutanolysin reduced its 180- to 200-kDa mass to 98 kDa without loss of enzymatic activity. The purified protease cleaved bovine gelatin, human placental type IV collagen, and the Aalpha chain of fibrinogen but not albumin, fibronectin, laminin, or myosin. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. Maximum production of the 98-kDa protease occurred during growth of S. gordonii CH1 in CDM containing 0.075% total amino acids at pH 7.0 with minimal aeration. Higher initial concentrations of amino acids prevented the release of the protease without reducing cell-associated enzyme levels, and the addition of an amino acid mixture to an actively secreting culture stopped further enzyme release. The purified protease was stored frozen at -20 degreesC for several months or heated at 50 degreesC for 10 min without loss of activity. These data indicate that S. gordonii produces an extracellular gelatinase/type IV collagenase during growth in medium containing minimal concentrations of free amino acids. Thus, the extracellular enzyme is a potential virulence factor in the amino acid-stringent, thrombotic, valvular lesions of bacterial endocarditis.
戈登氏链球菌是感染性细菌性心内膜炎的常见病因，但其毒力机制尚未完全明确。在本研究中，从化学限定培养基（CDM）的培养上清中回收链球菌蛋白酶，并通过硫酸铵沉淀、离子交换和凝胶过滤柱色谱法进行分离。通过它们在硫酸铵中的不同溶解度和对合成肽的特异性区分出三种蛋白酶。其中一种酶可切割胶原蛋白类似物甘氨酰-脯氨酰-4-甲氧基-β-萘酰胺、2-呋喃丙烯酰-亮氨酰-甘氨酰-脯氨酰-丙氨酸（FALGPA）和对苯基偶氮苄氧羰基-脯氨酰-亮氨酰-甘氨酰-脯氨酰-精氨酸（pZ-肽），并且在与肽聚糖片段结合时从链球菌中释放出来。用变溶菌素处理这种蛋白酶可使其180至200 kDa的分子量降至98 kDa而不丧失酶活性。纯化的蛋白酶可切割牛明胶、人胎盘IV型胶原蛋白和纤维蛋白原的Aα链，但不能切割白蛋白、纤连蛋白、层粘连蛋白或肌球蛋白。酶活性受到苯甲基磺酰氟的抑制，表明它是一种丝氨酸型蛋白酶。在含有0.075%总氨基酸、pH 7.0且通气量最小的CDM中培养戈登氏链球菌CH1期间，98 kDa蛋白酶的产量最高。较高的初始氨基酸浓度可阻止蛋白酶的释放，而不降低细胞相关酶水平，并且向活跃分泌的培养物中添加氨基酸混合物会停止进一步的酶释放。纯化的蛋白酶在-20℃冷冻保存数月或在50℃加热10分钟后活性均无损失。这些数据表明，戈登氏链球菌在含有最低浓度游离氨基酸的培养基中生长期间会产生一种细胞外明胶酶/IV型胶原酶。因此，这种细胞外酶是细菌性心内膜炎氨基酸缺乏、血栓形成的瓣膜病变中的一种潜在毒力因子。
