A method for characterizing the thermal stability and antimicrobial binding to Lipopolysaccharides of Gram-negative isogenic mutant strains

Belén Navarro; Mackarenna Alarcón; Maricarmen Osees; Felipe Gómez-Alvear; Romina V Sepúlveda; Jaime Huerta; María Cecilia Opazo; Daniel Aguayo

doi:10.1016/j.mex.2021.101474

A method for characterizing the thermal stability and antimicrobial binding to Lipopolysaccharides of Gram-negative isogenic mutant strains

MethodsX. 2021 Jul 30:8:101474. doi: 10.1016/j.mex.2021.101474. eCollection 2021.

Authors

Belén Navarro¹, Mackarenna Alarcón¹, Maricarmen Osees¹, Felipe Gómez-Alvear¹, Romina V Sepúlveda¹, Jaime Huerta¹, María Cecilia Opazo^{2

3}, Daniel Aguayo^{1

4}

Affiliations

¹ Center for Bioinformatics and Integrative Biology (CBIB), Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago 8370146, Chile.
² Instituto de Ciencias Naturales, Facultad de Medicina Veterinaria y Agronomía, Universidad de las Américas, Santiago, Chile.
³ Millennium Institute of Immunology and Immunotherapy.
⁴ Interdisciplinary Center for Neuroscience of Valparaíso, Faculty of Science, University of Valparaíso, Valparaíso 2340000, Chile.

Abstract

Isothermal Titration Calorimetry (ITC) is widely employed to assess antimicrobial affinity for lipopolysaccharide (LPS); nevertheless, experiments are usually limited to commercially available-LPS chemotypes. Herein we show a method that uses Differential Scanning Calorimetry (DSC) to characterize homogeneity artificial vesicles of LPS (LPS-V) extracted from isogenic mutant bacterial strains before analyzing the antimicrobial binding by ITC. This method allows us to characterize the differences in the Polymyxin-B binding and gel to crystalline liquid (β↔α) phase profiles of LPS-V made of LPS extracted from Escherichia coli isogenic mutant strains for the LPS biosynthesis pathway, allowing us to obtain the comparable data required for new antimicrobial discovery. A method for:•Obtaining LPS vesicles from isogenic mutant bacterial strains.•Characterize artificial LPS vesicles homogeneity.•Characterize antimicrobial binding to LPS.

Keywords: Antimicrobial binding; Differential Scanning Calorimetry (ITC); Isothermal Titration Calorimetry (ITC); Lipopolysaccharides.