Effect of Staphyloxanthin on Biophysical Properties of Membrane Models: A Spectroscopy Study Jessica Múnera-Jaramillo1, Gerson-Dirceu López2,3, Elizabeth Suesca3, Chad Leidy3 , Marcela Manrique-Moreno1* 1 Chemistry Institute, Faculty of Exact and Natural Sciences, University of Antioquia, Medellin, Colombia. 2 Laboratory of Advanced Analytical Techniques in Natural Products (LATNAP), Chemistry Department, Universidad de los Andes, Bogotá D.C., Colombia. 3 Biophysics Group, Department of Physics, Universidad de los Andes, Bogotá D.C., Colombia. e-mail: marcela.manrique@udea.edu.co Dried-freeze cells were dissolved in MeOH (BHT 0.1%) Centrifugation and extraction of pigment Combination of all methanolic phases Collection of organic phases Centrifugation and extracts collection Drying and concentration Extract purification by using PTCL STX identification by using HPLC-MS 1 Extraction and Identification of Staphyloxanthin Anisotropy and GP measurements Cell wall Cell membrane Cytoplasm S. aureus For the S. aureus cell membrane, PG and CL appear to be the main phospholipids CL DMPG STX Transition temperature (Tm) analysis by FT-IR Lipids System/ DMPG CL DMPG:CL Staphyloxanthin (mol %) 0 22.9 43.4 28.7 5 22.5 42.6 29.2 10 21.3 42.4 29.0 15 20.0 42.1 27.8 20 21.0 42.0 27.0 Figure 3. Peak positions of the νCH2 vibrations bands of the methylene groups as functions of temperature in the presence of different concentrations of STX for (A) DMPG, (B) CL, (C) DMPG:CL (80:20) systems. Table 1. Phase transition (Tm) temperatures, of the supported bilayers of DMPG, CL, and DMPG:CL (80:20) by FTIR. Standard deviations are ≤0.1 °C. • Increasing concentrations of STX decrease the Tm values, especially for systems that include DMPG • Presumably, the STX modifies the packing of hydrocarbon chains of phospholipids, which results in a phase gel disruption Transition temperature analysis by FT-IR DMPG CL STX MLVs from stock solutions of DMPG:CL in CHCl3. The systems included DPH or Laurdan probe and STX (5, 10, 15, 20%) Measurements on PC-1 ISS spectrofluorometer. Temperature range: 10-45 °C Symmetric stretching vibration (2970-2820 cm-1), Tensor II spectrometer, BioATR II unit DMPG CL STX Supported lipid bilayers (SLBs) from stock solutions CHCl3 (DMPG:CL (80:20) + STX (1, 5, 10, 15, 20%) Hydration with buffer and peptide above the Tm of systems Staphylococcus aureus (S. aureus) is a Gram-positive bacteria considered one of the most frequent pathogens in hospitals.1 One of the most interesting features of S. aureus is the synthesis of lipids as a response to external factors.2 Recently, staphyloxanthin (STX) production has been associated with bacteria pathogenicity since it is related to a higher tolerance to oxidative stress and presumably it plays a role in regulating membrane properties.3, 4 In this study was evaluated the effect of increasing concentrations of STX on the thermotropic properties of cell membrane models of S. aureus. Infrared spectroscopy and fluorescence spectroscopy were used to evaluate the effect on system phase-transition temperature (Tm), and the changes in fluidity, respectively. (1) Lee, A. S., et al. Nature reviews Disease primers, 4(1), 1-23, 2018. (2) Braungardt, et al. BioMed research international, 2019. (3) Manrique-Moreno, M., et al. Membranes, 12(10), 945, 2022. (4) Xue, L., et al. Infection and Drug Resistance, 2151-2160. 2019. Acknowledges: Minciencias Grant 120480763040, RC 846-2018 and University of Antioquia (Vicerectoria de Investigaciones) Introduction Results and Discussion Methodology STX is the main compound in the S. aureus total carotenoids. That molecule protects the microorganism from oxidative damage. Additionally, it modulates the cell membrane's physical properties, such as fluidity • The incorporation of bacterial pigment STX on representative lipid models of S. aureus cell membranes influences the physical states of the systems. • The gradual inclusion of STX in SLBs depresses the lipid phase transition through the organized phase disruption. • Variation in anisotropy values as a function of the amount of STX shows that the pigment induces a strong effect in the hydrophobic core of the lipid systems. • STX induces the condensation of polar groups in the liquid phase, evidenced by the change in GP values for representative membrane systems. Conclusions References EBSA Congress 30th July - 4th August, 2023, Stockholm, Sweden 5 10 15 20 25 30 35 40 45 50 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 A n is o tr o p y Temperature (°C) DMPG:CL DMPG:CL+ STX 5% DMPG:CL+ STX 10% DMPG:CL+ STX 15% DMPG:CL+ STX 20% Figure 2. Thermotropic measurements of Laurdan Generalized Polarization as temperature function Anisotropy measurements GP Measurements Figure 1. Thermotropic measurements of Anisotropy as temperature function More organized system Less hydrated system • The increment in concentrations of STX increases the lipid ordering in both liquid- crystalline and gel phases • Systems hydration is slightly affected in the gel phase, while significant variations occurred in the liquid phase in increasing STX 3 2 5 10 15 20 25 30 35 40 45 50 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 G en er al iz ed P o la ri za ti o n Temperature (°C) DMPG:CL DMPG:CL+ STX 5% DMPG:CL+ STX 10% DMPG:CL+ STX 15% DMPG:CL+ STX 20% 10 20 30 40 50 2849.5 2850.0 2850.5 2851.0 2851.5 2852.0 2852.5 2853.0 2853.5 W a v e n u m b e r (c m -1 ) Temperature (°C) DMPG:CL DMPG:CL + STX 5% DMPG:CL + STX 10% DMPG:CL + STX 15% DMPG:CL + STX 20% (C) 25 30 35 40 45 50 2849.5 2850.0 2850.5 2851.0 2851.5 2852.0 2852.5 2853.0 2853.5 W av en u m b er ( cm -1 ) Temperature (°C) CL CL+STX 5% CL+STX 10% CL+STX 15% CL+STX 20% (B) 10 15 20 25 30 35 2849.5 2850.0 2850.5 2851.0 2851.5 2852.0 2852.5 2853.0 2853.5 W av en u m b er ( cm -1 ) Temperature (°C) DMPG DMPG+ STX 5% DMPG+ STX 10% DMPG+ STX 15% DMPG+ STX 20% (A)