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Dissertation
Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin
Doctor of Philosophy (Ph.D.), Drexel University
Nov 2003
DOI:
https://doi.org/10.17918/00009328
Abstract
We characterized the expression of a putative toxin of B. anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O (LLO), perfringolysin O (PFO) and streptolysin O (SLO). We have named the 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 BHI broth, or in other rich bacteriologic media, but 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 stationary phase. These observations are supported, in part, by semi-quantitative 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 activities 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 growth medium. When strain UT231 was complimented 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 E. coli was extremely active against washed human erythrocytes, with complete hemolysis detected at ~30 molecules of rALO per erythrocyte. We have gone on to examine the interaction of the UT231 strain with mouse and human macrophage-like cell lines. These experiments demonstrate that ALO does not appear to be required for escape of B. anthracis from these cells.
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Details
- Title
- Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin
- Creators
- Jeffrey G. Shannon
- Contributors
- Richard F. Rest (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- [iii], vii, ii, iv, 114 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Medicine; Drexel University
- Other Identifier
- 991021889061304721