Role of Tryptophan Residues in the Toxicity and Photosensitized Inactivation of Escherichia coli α-Hemolysin

REID, LARA O.; THOMAS, ANDRÉS H.; HERLAX, VANESA; DÁNTOLA, M. LAURA

BIOCHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2020 vol. 59 p. 4213 - 4224

0006-2960

- Hemolysin (HlyA) is an extracellular protein toxin secreted by uropathogenic strains of Escherichia coli that inserts into membranes of eukaryotic cells. The main goal of the present work was to investigate the involvement of tryptophan (W) residues in the hemolytic activity of HlyA. We investigated the hemolytic activity of six single point mutant proteins, where one of the four Ws was replaced by cystein (C) or leucine (L). We also, analyzed the photoinactivation of HlyA with pterin (Ptr), an endogenous photosensitizer, as a method of unspecific oxidation of W and tyrosine (Y) residues. HlyA photoinactivation was analyzed by UV-Vis spectrophotometry, hemolytic activity measurement, fluorescence spectroscopy and electrophoretic analysis. Results indicate that Ws are important in the hemolytic process. Specifically the chemical structure of the amino acid at position 578 is important for the acylation process of HlyA at residue K563. Furthermore, the exposure of HlyA to UV radiation, with energy similar to that experienced under sun exposition, in the presence of Ptr induces the inactivation of the toxin, causing chemical changes in, at least, W and Y, being the rate of damage of W residues faster than the one observed for Y residues. This work not only deepens into the structure-function relationship of the toxin, but also introduces the possibility of using photoinactivation of HlyA for potential applications, such as the obtaining of innocuous molecules for vaccine production and the elimination of the toxin from contaminated surfaces and drinking water.