Plotted are the percent of detectible mRNA species (Y-axis) with a half life of ≤2.5, 5, 15, 30, or >30 min during exponential- and/or stationary- phase growth (X-axis).

(A) Representative screening effort results; dark blue arrow indicates substrate alone (negative control); grey arrow indicates enzyme (positive control); light-blue arrows indicate compounds that inhibited RnpA activity by ≥50%. (B) An agarose gel-based assay was used to distinguish bona-fide RnpA inhibitors from primary screening artifacts. Shown is the gel mobility of molecular weight marker, spa mRNA in the absence (−) or presence (+) of 20 pmol RnpA and RnpA-mediated spa mRNA degradation in the presence of increasing concentrations of RNPA1000, as described in Materials and Methods. (C) Structure of RnpA-inhibitory molecule RNPA1000.

Plotted are all GeneChip detected transcript levels at 0 and 5 min post-transcriptional arrest. Red, blue, and yellow data points represent all GeneChip measurable transcripts that were considered present, present in one sample but absent in second, or absent in both RNA samples, respectively. Grey dashed line indicates the lower limit of sensitivity for each sample. (A) A comparison of the mRNA levels of DMSO treated cells; 90% of all transcripts decrease ≥2-fold at 5 min post-transcriptional arrest (Left Panel). A comparison of the mRNA levels of cells challenged with sub-inhibitory concentration of RnpA-inhibitor; addition of the agent resulted in pronounced stabilization of S. aureus transcripts (Right Panel). (B) Microtiter plate assay illustrating the in vitro antimicrobial effects of indicated concentration of RNPA1000 (across top) against S. aureus RN4220 pRNPA-A.S. (RnpA depleted cells; top panel), RN4220 pRNPA (RnpA overexpressor cells; center panel) and RN4220 pCN51 (vector; bottom panel) when grown in the presence of 2.5 µM CdCl₂. All strains were assessed twice; arrows indicate MIC values.

(A) SDS-PAGE of purified recombinant S. aureus RnpA; shown are molecular markers (Lanes M), 2.5 µg and 25 µg elution products (Lanes 1 and 2, respectively). The band at ∼17.2 kDa (solid arrow; Band 2) was confirmed to be S. aureus RnpA by tandem mass spectrometry (Wistar Institute; Philadelphia, PA), whereas top-hits for minor contaminants (dashed arrows) were determined to be E. coli 50S ribosomal protein L3 (Band 1) or S. aureus RnpA polypeptide fragments, corresponding to amino acids 11–107 (Bands 3 and 4) or 12–107 (Band 5). (B) Gel-mobility of 1 µg of total S. aureus RNA following 60 min incubation in the absence (−) or presence (+) of 50 pmol of each putative ribonuclease (indicated) in 1X reaction buffer (2 mM NaCl, 2 mM MgCl₂, 50 mM Tris-HCl, pH 6.0). Recombinant S. aureus RnpA catalyzed RNA digestion; equivalent amounts of other putative S. aureus ribonucleases demonstrated little or no ribonuclease activity. (C) Mobility of 0.5 pmol in vitro transcribed spa mRNA following 60 min incubation in the absence (0 pmol) or presence of the indicated amount of RnpA protein in 1X reaction buffer. Recombinant S. aureus RnpA catalyzed digestion of spa mRNA; molecular weight markers (M) are shown. (D) Plotted are measurements for all mRNA species measured on a GeneChip at 0 (X-axis) and 10 min (Y-axis) post-transcriptional arrest. Red data points represent all transcripts that are considered present by Affymetrix algorithms at both 0 and 10 min following transcriptional arrest. Blue data points represent transcripts that were considered present in one sample but not detectable in the second. Yellow data points represent transcripts that are not expressed/detectible in either RNA sample. Comparisons of the transcript titer of a given data point at 0 min to that of 10 min post-transcriptional arrest serves as a measurement of the degradation of that particular mRNA species. Grey dashed line indicates the lower limit of sensitivity for each sample. A total of 88% and 87% of all transcripts produced in cells harboring vector (pCN51; left panel) or overexpressing RnpA (pRNPA; middle panel), respectively, exhibit a decrease in transcript titer of ≥2-fold following 10 min transcriptional arrest. Conversely, many RNA species are stabilized in RnpA depleted cells (pRNPA-A.S.; right panel). Western blotting confirmed the amount of S. aureus RnpA protein present in each strain (Supplemental Figure S1B.).

(A) MTT-cytotoxicity assay results of HepG2 cells exposed to compound solvent (DMSO; negative control), Mitomycin C (positive control), and indicated amount of RNPA1000. (B) Shown are the average daily (X-axis) percent surviving animals (Y-axis) following no treatment (closed diamonds; negative control), vancomycin treatment (closed squares; 1 mg/kg; positive control), or RNPA1000 treatment; 64 mg/kg (open circles); 128 mg/kg (open squares), 256 mg/kg (open triangles). Experiment was repeated twice; replicate measurements are shown in Supplemental Table S3. (C) In vitro biofilm assay results; graphed are the number of catheter-associated S. aureus following 1, 2, or 3 days of no antimicrobial treatment (untreated; UT) or exposure to 5, 10, or 20 times the MIC for RNPA1000. Boxes define the interval between the 25^th and 75^th percentile. Bars extending upward indicate the boundary defined by the value 1.5X higher than the 75^th percentile while those extending downward indicate the boundary defined by a value 1.5X lower than the 25^th percentile. Filled circles indicate individual values outside these two extremes.