Shannon Jeffrey G, Ross Cana L, Koehler Theresa M, Rest Richard F
Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.
Infect Immun. 2003 Jun;71(6):3183-9. doi: 10.1128/IAI.71.6.3183-3189.2003.
We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). Although B. anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR of alo mRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which the alo gene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented in trans with native alo on a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain of B. anthracis in rich media in vitro. To further support the alo gene product being a hemolysin, recombinant B. anthracis ALO (rALO) purified from Escherichia coli was extremely active against washed human erythrocytes, with complete hemolysis detected at approximately 30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.
我们对炭疽芽孢杆菌的一种假定毒素的表达进行了表征，该毒素是胆固醇依赖细胞溶素（CDC）家族的成员，该家族包括李斯特菌溶素O、产气荚膜梭菌溶素O和链球菌溶素O。我们将这种细胞毒素命名为炭疽溶素O（ALO）。尽管炭疽芽孢杆菌在临床环境中表现出最小的溶血活性，但我们发现，斯特恩菌株7702在脑心浸液肉汤或其他丰富的细菌培养基中生长时表现出溶血活性，但在Luria-Bertani（LB）肉汤中生长时分泌的溶血素量几乎检测不到。向LB肉汤中添加葡萄糖可增加分泌的溶血活性量。溶血活性在对数中期至后期达到最大值，在稳定期降低。这些观察结果部分得到了alo mRNA的半定量逆转录PCR的支持。生长上清液中的溶血活性在还原剂存在下增加，并且以剂量依赖的方式几乎完全被胆固醇抑制；这两种活性都是CDC毒素的特征。斯特恩菌株7702的一个突变体UT231，其中alo基因被删除并用卡那霉素盒取代，向生长培养基中分泌的溶血活性几乎检测不到。当菌株UT231用低拷贝数质粒上的天然alo进行反式互补时[菌株UT231（pUTE554）]，它恢复了分泌溶血活性的能力，表明ALO是该炭疽芽孢杆菌菌株在体外丰富培养基中分泌的主要溶血素。为了进一步支持alo基因产物是一种溶血素，从大肠杆菌中纯化的重组炭疽芽孢杆菌ALO（rALO）对洗涤过的人红细胞具有极强的活性，在每个红细胞约30个rALO分子时检测到完全溶血。考虑到CDCs对其他革兰氏阳性菌的毒力作用，我们推测ALO可能在炭疽毒力中起作用。
