Mechanistic insights into the interactions between S. aureus amyloids and cell membranes L. Bonnecaze, K. Jumel, L. Khemtemourian, S. Lecomte, C. Feuillie, M. Molinari, M. Mathelié-Guinlet* Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, F-33600 Pessac, France S. aureus PSMs An opportunistic pathogen Antibiotic multiresistance PSMα3, an amyloid-like cytotoxic entity (f-)MEFVAKLFKFFKDLLGKFLGNN  Cross-α fibrillation Misunderstood interactions with both eukaryotic and bacterial membranes & Unknown influence of N-formylation, in toxic processes • Cytotoxicity F3A mutant No fibrillation reduced cytotoxicity but gained antibacterial activity f-WT thick clustered fibers f-F3A amorphous aggregates PSMα3 fibrillation [1] Peschel et Otto, Nat Rev Microbiol., 2013 [2] Tayeb-Fligelman et al., Science, 2017 [3] Malishev et al., J.Mol.Biol., 2018 *marion.mathelie-guinlet@u-bordeaux.fr [2] At 10 µM, both peptides induce only limited damage to PC-membranes f-F3A acts preferentially on bacterial membranes Impact of PSMα3 on biomimetic membranes Nanoscale visualization of PSMα3 membrane activity Accumulation and elongation of fibrils on PC-membranes, only on fluid phases Formation and propagation of a « front » and eventually holes around the growing fibrils On PE / PG membranes, peptides do not deposit nor disrupt the SLB No lipid-dependent changes in 2nd structure for both peptides high α-helical content conserved (>50%) DOPE/DOPG + f-WT / f-F3A 5µM 120’ 0’ 600nm T cells Gram + 0 20 40 60 80 100 Toxicity (%) WT F3A 1. Incubation 3days @ 37°C in buffer (ThT fluorescence) 2. Interactions with supported lipid bilayers (AFM & ATR-FTIR) • Inflammation • Biofilm Is toxicity driven by fibrils? Eukaryotic membranes PC vs. Bacterial membranes PE/PG Buffer : 10 mM sodium phosphate buffer + 150 mM NaCl, pH 8,0 SLB formed by SUV fusion Methods [2-3] [1] f At 5-10 µM, f-WT and f-F3A induce only small damage on eukaryotic membranes whereas f-F3A disrupts bacterial membranes 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 f-WT f-F3A Buffer ThT fluorescence intensity (x103) Time (h) 200nm 200nm 0 250 500 750 1000 -1.0 0.0 1.0 2.0 3.0 4.0 Distance (nm) Height (nm) 60’ 120’ 600nm DOPC DPPC 0’ DOPC/DPPC + f-WT 5µM 15’ 30’ 600nm 0 150 300 450 600 −0.5 0.0 0.5 1.0 1.5 Height (nm) Distance (nm) 200nm 200nm 0’ DOPC/SM/Chol + f-F3A 5µM 10nm 140MPa Elasticity Elasticity 1500 1550 1600 1650 1700 0.0 0.2 0.4 0.6 0.8 1.0 Absorbance (u.a.) Wavenumber (cm-1) f-WT spectrum Fit spectrum Turn Alpha Random // Beta DOPC DOPC/DPPC (1:1) DOPC/SM/Chol (67:8:25) DOPE/DOPG (1:1) 0.6 0.9 1.2 1.5 1.8 2.1 2.4 fWT10µM 3h fF3A 10µM 3h Dichroic ratio DOPC DOPC/DPPC (1:1) DOPC/SM/Chol (67:8:25) DOPE/DOPG (1:1) 0 20 40 60 80 100 120 140 Lipid coverage [p-pol] (%) + f-WT 10µM, 3h + f-F3A 10µM, 3h SLB Accumulation on membranes At high concentrations (50µM), f-WT disrupts PC-membranes while f-F3A rather destroys bacterial PE/PG ones. PC/SM/Chol PE/PG 0 20 40 60 80 100 Lipid coverage (%) + f-WT 50µM + f-F3A 50µM At the nanoscale, hydrophobic effects likely drive PSMα3 accumulation and fiber elongation at the membrane interface Funded by the European Union (EU). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the EU. Neither the EU nor the granting authority can be held responsible for them.