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Infection and Immunity, June 2003, p. 3183-3189, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3183-3189.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of Anthrolysin O, the Bacillus anthracis Cholesterol-Dependent Cytolysin

Jeffrey G. Shannon,1 Cana L. Ross,2 Theresa M. Koehler,2 and Richard F. Rest1*

Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129,1 Department of Microbiology and Molecular Genetics, University of Texas-Houston Medical School, Houston, Texas 770302

Received 18 October 2002/ Returned for modification 3 December 2002/ Accepted 13 February 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 ~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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Ln., Philadelphia, PA 19129. Phone: (215) 991-8382. Fax: (215) 848-2271. E-mail: rickrest{at}drexel.edu.

Editor: D. L. Burns

Infection and Immunity, June 2003, p. 3183-3189, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3183-3189.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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