Biochemical and physiological characteristics of HlyA, a pore-forming cytolysin of Vibrio cholerae serogroup O1.
Among the various toxins produced by the bacterial species Vibrio cholerae is HlyA, a cytolytic protein commonly called the E1 Tor hemolysin. HlyA is synthesized and processed in a complex manner involving various processed or degraded forms, that may co-purify and complicate the interpretation of biochemical and physiological experiments. In this study a single form of HlyA was purified by gel filtration and chromatofocusing using fast protein liquid chromatography in the presence of protease inhibitors. A 45-fold purification was obtained, with a final recovery of 17% of pure 60,000 mol. wt HlyA. A significant improvement in specific activity to 8.5 x 10(6) Chinese hamster ovary tissue culture units per mg protein was obtained. Physiological activity studies indicated that cytolysis of erythrocytes (hemolysis) was inhibited by oxygen: storage of HlyA under oil, and experimentation in N2-flushed buffers maintained activity. HlyA-mediated lysis of human erythrocytes was characterized by a significant lag phase, followed by a rapid induction of hemolysis. Hemolysis was inhibited by sucrose, an osmotic protectant, suggesting that the initial action of HlyA on erythrocytes is to raise the basal cation permeability of the cell membrane. The most likely cytolytic mechanism is thus the formation of transmembrane lesions such as homopolymer pores in target cells, as has been found for toxins from numerous other bacterial pathogens.
Sathyamoorthy
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Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, DC 20204, USA.
Huntley
J S
JS
Hall
A C
AC
Hall
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D016466:CHO Cells; D006224:Cricetinae; D004355:Drug Stability; D004912:Erythrocytes / Q000187:drug effects*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D006461:Hemolysis; D006801:Humans; D008969:Molecular Sequence Data; D014734:Vibrio cholerae / Q000737:chemistry*
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; C084287:hlyA protein, Vibrio cholerae