Fig 2.

SaeRS is required for Nuc enzyme production. Protein immunoblot assays for Hla (A) and Nuc (B) were performed with spent medium from WT LAC and strains containing hla, nuc, sae, and agr mutations grown to stationary phase. The long and processed forms of Nuc are designated NucB and NucA, respectively. A complemented (Comp) version of each mutant was included as a control. (C) The Nuc enzyme activity of these samples was determined with a FRET assay. Statistical analyses were performed with a two-tailed Student t test. (D) Nuc immunoblot assays of sae and agr mutants of strains LAC, MW2, UAMS-1, Newman, and COL were performed, and the NucA and NucB bands are indicated. Variation in Nuc protein levels across S. aureus isolates is SaeRS dependent. (E) Nuc activity in vivo following intraperitoneal infection with S. aureus. Mice were infected with 2 × 10⁷ CFU of WT USA300 or isogenic agr, sae, and nuc mutants. Nuc activity in peritoneal fluids was assayed at 8 h postinfection with a FRET assay (n = 5 mice per strain for the WT and the agr and sae mutants and 4 mice for the nuc mutant). P values were determined by paired t test. PBS, phosphate-buffered saline. The inset shows the number of CFU recovered from mice at the time of peritoneal fluid harvest. ***, P = 0.001 (determined by paired t test).