Li Jihong, McClane Bruce A
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Appl Environ Microbiol. 2014 Mar;80(5):1701-9. doi: 10.1128/AEM.03440-13. Epub 2013 Dec 27.
Clostridium perfringens causes histotoxic infections and diseases originating in animal or human intestines. A prolific toxin producer, this bacterium also produces numerous enzymes, including sialidases, that may facilitate infection. C. perfringens type D strain CN3718 carries genes encoding three sialidases, including two large secreted sialidases (named NanI and NanJ) and one small sialidase (named NanH) that has an intracellular location in log-phase cultures but is present in supernatants of death phase cultures. Using isogenic mutants of CN3718 that are capable of expressing only NanJ, NanI, or NanH, the current study characterized the properties and activities of each sialidase. The optimal temperature determined for NanJ or NanH enzymatic activity was 37°C or 43°C, respectively, while NanI activity increased until temperature reached 48°C. NanI activity was also the most resistant against higher temperatures. All three sialidases showed optimal activities at pH 5.5. Compared to NanJ or NanH, NanI contributed most to the sialidase activity in CN3718 culture supernatants, regardless of the substrate sialic acid linkage; NanI also released the most sialic acid from Caco-2 cells. Only NanI activity was enhanced by trypsin pretreatment and then only for substrates with an α-2,3- or α-2,6-sialic acid linkage. NanJ and NanI activities were more sensitive than NanH activity to two sialidase inhibitors (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid and siastatin B). The activities of the three sialidases were affected differently by several metal ions. These results indicated that each C. perfringens sialidase has distinct properties, which may allow these enzymes to play different roles depending upon environmental conditions.
产气荚膜梭菌可引发源于动物或人类肠道的组织毒性感染和疾病。这种细菌是一种多产毒素的细菌，还能产生多种酶，包括唾液酸酶，这些酶可能会促进感染。D型产气荚膜梭菌菌株CN3718携带编码三种唾液酸酶的基因，其中包括两种大型分泌型唾液酸酶（分别命名为NanI和NanJ）和一种小型唾液酸酶（命名为NanH），后者在对数期培养物中定位于细胞内，但在死亡期培养物的上清液中存在。本研究利用能够仅表达NanJ、NanI或NanH的CN3718同基因突变体，对每种唾液酸酶的特性和活性进行了表征。确定NanJ或NanH酶活性的最佳温度分别为37°C或43°C，而NanI的活性在温度达到48°C之前一直增加。NanI的活性对较高温度也最具抗性。所有三种唾液酸酶在pH 5.5时均表现出最佳活性。与NanJ或NanH相比，无论底物唾液酸的连接方式如何，NanI对CN3718培养上清液中的唾液酸酶活性贡献最大；NanI从Caco-2细胞中释放的唾液酸也最多。只有NanI的活性通过胰蛋白酶预处理得到增强，并且仅对于具有α-2,3-或α-2,6-唾液酸连接的底物。NanJ和NanI的活性比NanH的活性对两种唾液酸酶抑制剂（N-乙酰-2,3-脱氢-2-脱氧神经氨酸和西他司丁B）更敏感。几种金属离子对三种唾液酸酶活性的影响各不相同。这些结果表明，每种产气荚膜梭菌唾液酸酶都具有独特的特性，这可能使这些酶根据环境条件发挥不同的作用。
