title: Brief heat treatment causes a structural change and enhances cytotoxicity of the Escherichia coli α-hemolysin.
abstract: α-Hemolysin (HLY) is an important virulence factor for uropathogenic Escherichia coli. HLY is a member of the RTX family of exotoxins secreted by a number of Gram-negative bacteria. Recently, it was reported that a related RTX toxin, the Mannheimia haemolytica leukotoxin, exhibits increased cytotoxicity following brief heat treatment. In this article, we show that brief heat treatment (1 min at 100°C) increases cytotoxicity of HLY for human bladder cells, kidney epithelial cells (A498) and neutrophils. Heat treatment also increased hemolysis of human red blood cells (RBCs). Furthermore, heat treatment of previously inactived HLY restored its cytotoxicity. Heat-activated and native HLY both required glycophorin A to lyse RBCs. Native and heat-activated HLY appeared to bind equally well to the surface of A498 cells; although, Western blot analyses demonstrated binding to different proteins on the surface. Confocal microscopy revealed that heat-activated HLY bound more extensively to internal structures of permeabilized A498 cells than did native HLY. Several lines of spectroscopic evidence demonstrate irreversible changes in the structure of heat activated compared to native HLY. We show changes in secondary structure, increased exposure of tryptophan residues to the aqueous environment, an increase in molecular dimension and an increase in hydrophobic surface area. These properties are among the most common characteristics described for the molten globule state, first identified as an intermediate in protein folding. We hypothesize that brief heat treatment of HLY causes a conformational change leading to significant differences in protein-protein interactions that result in increased cytotoxicity for target cells.
authors.affiliation: Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Aulik
authors.forename: Dhammika N
authors.initials: DN
authors.lastname: Atapattu
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Czuprynski
authors.forename: Darrel R
authors.initials: DR
authors.lastname: McCaslin
mesh_terms: D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005260:Female; D006021:Glycophorins / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism*; D006461:Hemolysis / Q000502:physiology; D006358:Hot Temperature; D006801:Humans; D056004:Molecular Dynamics Simulation; D009504:Neutrophils / Q000378:metabolism; D017510:Protein Folding; D014364:Tryptophan / Q000378:metabolism
chemical_list: D006021:Glycophorins; D006460:Hemolysin Proteins; D014364:Tryptophan