Fig 6.

Hsp104^A503S disrupts S. aureus biofilm formation. (A) Schematic depicting the experimental design was created using BioRender.com. (B) SH1000 WT or SH1000 Δαβpsm S. aureus was incubated in 96 well plates for 24 h to allow biofilm biomass to accumulate. Following washing, residual biomass was stained with crystal violet dye to visualize biomass. Images (top) show wells that are all from a single trial. Wells were then resuspended and quantified. Biomass for each condition was compared to the treatment of SH1000 S. aureus control treated with buffer using a two-tailed, unpaired t-test. (N ≥ 6 biological replicate trials, with three technical replicate wells for each trial, bars show mean ± SEM, **P < 0.01, ****P < 0.0001, and ns P > 0.05). (C) Experiments were performed as in B using SH1000 WT S. aureus. Here, following washing, residual biomass was treated with buffer, Hsp104^WT, Hsp104^A503S, or Hsp104^DPLA-DWB plus or minus Hsp40 and Hsp70, as indicated, for 24 h at 37°C. Wells were then stained with crystal violet to visualize biomass. Images shown depict wells that are all from a single trial (top). Wells from B were resuspended and quantified (bottom). Biomass for each condition was compared to control treatment with buffer using a two-tailed, unpaired t-test. (N ≥ 4 biological replicate trials, with two technical replicate wells for each trial, bars show mean ± SEM, *P < 0.05, ****P < 0.0001, and ns P > 0.05). (D) Experiments were performed as in C using SH1000 Δαβpsm S. aureus. Following treatment, wells were stained with crystal violet to visualize biomass (top) and then resuspended and quantified (bottom). Biomass for each condition was compared to control treatment with buffer using a two-tailed, unpaired t-test. (N ≥ 4 biological replicate trials, with three technical replicate wells for each trial, bars show mean ± SEM, and all P values were greater than 0.05).