Growth of Staphylococcus aureus in the presence of oleic acid shifts the glycolipid fatty acid profile and increases resistance to antimicrobial peptides

Abstract

Staphylococcus aureus readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, S. aureus presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility. When S. aureus is grown conventionally, its membrane lipids contain a mix of branched-chain and straight-chain saturated fatty acids. However, when unsaturated fatty acids are present in the growth medium, they become a major part of the total fatty acid composition. This study explores the biophysical effects of incorporating straight-chain unsaturated fatty acids into S. aureus membrane lipids. Membrane preparations from cultures supplemented with oleic acid showed more complex differential scanning calorimetry scans than those grown in tryptic soy broth alone. When grown in the presence of oleic acid, the cultures exhibited a transition significantly above the growth temperature, attributed to the presence of glycolipids with long-chain fatty acids causing acyl chain packing frustration within the bilayer. Functional aspects of the membrane were assessed by studying the kinetics of dye release from unilamellar vesicles induced by the antimicrobial peptide mastoparan X. Dye release was slower from liposomes prepared from cells grown in oleic acid-supplemented cultures, suggesting that changes in membrane lipid composition and biophysics protect the cell membrane against peptide-induced lysis. These findings underscore the intricate relationship between the growth environment, membrane lipid composition, and the physical properties of the bacterial membrane, which should be considered when developing new strategies against S. aureus infections.

Figure 1.. DSC scans of S. aureus membrane and lipid preparations from cultures grown in TSB without supplementation.

Heating scans obtained from an aqueous suspension of membrane fragments (A). First heating scan, black trace; Second heating scan, red trace; third heating scan, blue trace. First heating scan of an aqueous suspension of MLVs made from whole lipid extracts (B). First heating scans of an aqueous suspension of MLVs (liposomes) made from the phospholipid and glycolipid fractions (C). Phospholipid fraction, black trace. Glycolipid fraction, red trace.

Figure 3.. DSC scans of S. aureus membrane preparations from cultures grown in TSB with oleic acid supplementation.

Heating scans obtained from an aqueous suspension of membrane fragments (A). First heating scan, black trace; Second heating scan, red trace; third heating scan, blue trace. First heating scan of an aqueous suspension of MLVs made from total lipid extracts (B). First heating scans of an aqueous suspension of MLVs made from the phospholipid and glycolipid fractions (C). Phospholipid fraction, black trace. Glycolipid fraction, red trace.

Figure 4.. Kinetics of CF release induced by the antimicrobial peptide mastoparan X from large unilamellar lipid vesicles made from total lipid extracts.

Aqueous peptide concentration was 5 μM in all experiments. Lipid phosphate concentration was 80 ± 20 μM. Representative dye release kinetics from liposomes based on lipid extracts from unsupplemented cultures (A), and from cultures supplemented with oleic acid (C). Room temperature, blue trace; 37 °C, red trace. Average τ of dye release using liposomes based on lipid extracts from unsupplemented cultures (B), and from cultures supplemented with oleic acid (D) at two temperatures. Room temperature, blue bars; 37 °C, red bars. Error bars represent the standard deviation from three independent samples.

Figure 5.. Diglucosyldiglyceride (DGDG) species distribution

in extracts obtained from cultures grown in TSB, blue bars and from cultures grown in TSB supplemented with oleic acid, orange bars. Data was obtained from Hines et al. (2020) [48].

Figure 6.

Artist’s representation of inverted lipid phases. Inverted hexagonal H_II (A) and an example of an inverted bicontinuous cubic phase (Im3m) (B). The drawings were generated using the Blender software [60].