Figure 3: Triple-acting fusions can eradicate intracellular S. aureus in murine osteoblasts.

(A) Intracellular eradication of GFP expressing S. aureus. Neonatal mouse whole calvaria were treated for 4 hours with chimeric PGHs or buffer alone, 24 h post inoculation with GFP expressing S. aureus. Calvaria were embedded in freeze media, sectioned, and subjected to fluorescence imaging. The image shown is a representative figure for triplicate sections of three separate calvaria. (B) Fluorescence intensity from these sections was measured in ImageJ and defined as arbitrary fluorescence standardized to the untreated control. Error bars represent SEM of four replicate experiments. (C) Ex-vivo intracellular S. aureus eradication. Infected calvaria were homogenized and CFU were counted post treatment. Error bars represent SEM. (D) Murine model of staphylococcal osteomyelitis. C57BL/6J mice were anesthetized and their femurs were surgically exposed. A trough was drilled through the bone cortex, and the damaged bone sites were inoculated with 1 × 10³ CFU S. aureus in agarose beads. After 24 hours, mice were treated (i.m. to site of infection) twice in a 24 h period with PBS or 5 mg/kg of triple fusion K-L or K-L-PTD1. The femurs were removed, homogenized, and plated to quantify the bacterial load. Bars indicate the average CFU recovered (N = 6). Both triple fusion K-L (p = 0.012) and K-L-PTD1 (p = 0.021) significantly reduced bacterial load as compared to no treatment, but the presence or absence of PTD1 had no significant effect (p = 0.73). Asterisks represent statistical significance as determined by one-way ANOVA followed by Tukey’s posthoc test.