FIGURE 5.

miR-143 and MMP-2 expression/activity in wounds fibroblasts is regulated by SA biofilm infection. Wound biopsies were collected at specified time points post-inoculation with S. aureus USA300 isogenic strains: USA300, USA300::rexB (ΔrexB) or USA300::sarA (ΔsarA). A, Representative, gelatin zymogram of d35 porcine infected burn wounds tissue. Based on molecular weight, pro- (70 kDa) and active MMP-2 (62 kDa) bands have been indicated. Quantification of active MMP-2 (62 kDa) band using densitometry. Data are mean ± SEM (n = 4) *P < 0.05 compared with ΔsarA. B, MMP-2 mRNA in d35 post-inoculation porcine infected burn wounds tissues quantified using real-time PCR. Data was normalized against 18S RNA as housekeeping gene. Data are mean ± SEM (n = 6) *P < 0.05 compared with ΔsarA. C, MMP-2 protein in d35 post-inoculation porcine infected burn wounds tissues quantified using ELISA. Data are mean ± SEM (n = 6). *P < 0.05 compared with ΔsarA. D, miR-143 expression in d35 post-inoculation porcine infected burn wounds tissues quantified using real-time PCR. Data was normalized against U6 snRNA as housekeeping genes. Data are mean ± SEM (n = 6) *P < 0.05 compared with ΔsarA. E-G, Human fibroblasts (hFb) were cultured with overnight conditioned media from SA biofilms. Mature biofilms developed on polycarbonate membrane in vitro were placed in cultured dish and incubated with fibroblast (Fb) culture media overnight. The conditioned media was harvested, centrifuged, filtered and then added to hFb. E, Schematic presentation of the experimental model. F, MMP-2 mRNA expression in hTERT immortalized cultured human fibroblasts (hFb) with conditioned media from biofilms cultured in vitro. Data are mean ± SEM (n = 6) *P < 0.05 compared with ΔsarA. G, miR-143 expression in hTERT immortalized cultured human fibroblasts (hFb) with conditioned media from biofilms cultured in vitro. Data are mean ± SEM (n = 3) *P < 0.05 compared with ΔsarA.