Figure 6. Small molecules that perturb flotillin oligomerization affect Rny activity in S. aureus.

(A) Galleria mellonella worms were infected by injection with various S. aureus strains (5 x 10⁶ cells); percent survival is shown. Control larvae were infected with saline solution. Larvae were incubated (37°C, 48 h) and surviving larvae counted. Data shown as mean ± SEM (n = 15 larvae/group; three independent experiments). Significance was measured by one-way ANOVA with Tukey’s comparison. ** p <0.01. Data shown as mean ± SD of three independent biological replicates (n = 3). (B) qRT-PCR analysis of rsaA and sau63 expression in WT, ΔfloA, ΔfloA+floA and Δrny mutants of S. aureus. Relative gene expression is normalized to WT levels. Significance was measured by one-way ANOVA with Tukey’s comparison. ** p <0.01. Data shown as mean ± SD of three independent biological replicates (n = 3). Each biological replicate included three technical replicates. (C) Molecular structure of flotillin-perturbing small molecules. (D) Immunodetection of flotillin in a fractioned sucrose gradient from the membrane fraction of cells treated with ZA, miltefosine and 5-DSA. FloA-containing protein complexes were detected using anti-FloA antibodies. (E) Immunodetection of Rny in DSM and DRM fractions of S. aureus cells treated with ZA, miltefosine and 5-DSA. Ampicilin-treated samples are negative control since ampicillin does not affect FMM.