Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba
Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba
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DOI：
10.1128/jcm.38.8.2858-2861.2000
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发表时间：
2000-08-01
影响因子：
9.4
通讯作者：
Paget, TA
中科院分区：
文献类型：
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作者：
Khan, NA;Jarroll, EL;Paget, TA
阿米巴角膜炎是一种由阿米巴属病原性物种引起的威胁视力的感染。虽然不是所有的阿米巴属都能引起角膜炎，但重要的是要区分致病菌种和非病原体。由于细胞外蛋白酶可能在眼部病理学中发挥作用，我们使用比色、细胞病变和酶谱测定来评估致病性和非致病性棘阿米巴的细胞外蛋白酶活性。比色测定，使用ate-linked蛋白质作为底物，显示在阿米巴条件培养基和分化的致病性和非致病性阿米巴胞外蛋白酶活性。将四孔板中的永生化角膜上皮细胞单层用于细胞病变效应（CPE）测定。致病性阿米巴分离株表现出显着的CPE永生化角膜上皮细胞，而非致病性菌株没有表现出CPE。用阿米巴条件培养基以及阿米巴加上皮细胞条件培养基进行蛋白酶酶谱分析。酶谱蛋白酶测定显示不同菌株的阿米巴不同的带型。在致病性阿米巴菌株，所有的蛋白酶带抑制苯甲基磺酰氟（PMSF），这表明丝氨酸型蛋白酶，而在非致病性菌株中，只有部分抑制观察到使用PMSF。在典型的实验室条件下生长，没有上皮细胞的致病性阿米巴菌株表现出一个共同的过度表达的蛋白酶带107 kDa的，这种蛋白酶没有观察到在非致病性阿米巴菌株。107-kDa蛋白酶在5至9.5的pH范围内表现出活性。
Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp, can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using ate-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with Acanthamoeba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic Acanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.