Fig. 3.

Unsaturated fatty acids decrease S. aureus membrane fluidity, resulting in enhanced EsxA expression. (A) USA300 cultures were grown in TSB alone or in TSB supplemented with the indicated fatty acids. The membrane fluidity of the bacteria was determined at various temperatures using the pyrene decanoic acid fluorescence method. (B) USA300 WT or fakA-mutant strains were grown in TSB alone or in TSB supplemented with 10 µM LA, and the membrane fluidity of the bacteria at 37 °C was determined as in A. The percent change in membrane fluidity with or without LA is plotted. (C) USA300 WT or fakA-mutant strains were grown at the indicated temperatures in TSB alone or in TSB supplemented with 10 µM LA. Whole-cell lysates were assessed for EsxA protein by Western blot. The fold increase in EsxA protein for each condition was plotted relative to the average EsxA protein for the indicated strain grown in TSB alone at 37 °C. (D) USA300 WT or pCrtMN strains were grown in TSB alone or in TSB supplemented with 10 µM LA. Whole-cell lysates were assessed for EsxA protein by Western blot. The fold increase in EsxA protein was plotted relative to the average EsxA protein level in the indicated strain grown in TSB alone. Significance was evaluated using the Mann–Whitney u test: **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.