Figure 1.
Solonamide B does not influence RNAIII expression in the presence of a constitutively active AgrC.
Cultures of RN10829 (P2-agrA; P3-blaZ) containing either pagrC-I-WT (A, denoted WT) or pEG11 (B, denoted Const.) were grown to OD600 0.4–0.5 where 1/10 a volume of AIP-containing supernatant and 5 µg/mL or 10 µg/mL Solonamide B (SolB) or DMSO was added. Controls not containing AIP were included to confirm the inducible and constitutive nature of reporters in A and B, respectively. Samples were obtained at various time points after addition and were analyzed for β-lactamase activity (P3 expression). A single representative experiment is depicted with bars representing the mean +/− standard error of the mean (SEM) from triplicate determinations of β-lactamase activity. Growth monitored for the WT strain confirms that the reduction in P3 expression is not due to growth impairment (C).
Figure 2.
Solonamide B competitively interferes with AIP binding to AgrC.
Agar plates containing the spa-lacZ reporter strain PC203 were supplied with 10 µl DMSO containing 0, 0.8, 4, 20, 100 and 500 µg Solonamide in DMSO and 20 µl of supernatant of overnight culture of 8325-4 (indicated as “WT”) or RN6911, Δagr mutant cells (indicated as “Δagr”) or TSB medium resulting in no (B) or the indicated concentrations of solonamide B (A, C, D).
Figure 3.
Assessment of competitive action of Solonamide B in P3-blaZ reporter strain.
The inhibitory activity of 5 µg/mL Solonamide B (SolB) on RNAIII expression controlled by wild type AgrC was assessed when challenged by increasing concentrations of AIP. β-lactamase activity was determined with and without Solonamide B for three independent cultures (#1–#3) 30 minutes after addition of Solonamide B and induction by AIP. Bars represent means +/− SEM from triplicate activity determinations (A). Average inhibition obtained by Solonamide B at increasing AIP concentrations. Bars represent the mean (+/− SEM) reduction in β-lactamase activity observed from the three biological replicates (B). P-values between AIP treatments were calculated by Student’s t-test (two-sided).
Figure 4.
Structures of AIP II and Solonamide B. AIP II (left), Solonamide B (right).
Figure 5.
Solonamide B inhibits agr-group I, II and IV.
The P3::lux reporter strains AN1 agr I, AN2 agr II, AN3 agrIII and agr IV AN4 were grown with DMSO (control), Solonamide B 5 µg/mL, and Solonamide B 10 µg/mL. Luminescence was recorded after 2½ hours of growth in the presence of the compound. The luminescence is scored in % luminescence relative to the control (DMSO) set to 100% for each agr group individually and standard deviations are calculated based on three replicates.
Figure 6.
Solonamide B specifically reduces α-hemolysin production.
1–256 fold dilutions of sterile filtered 24 hour culture supernatants from USA300 cultures grown with DMSO, 5 µg/mL or 10 µg/mL of Solonamide B, were tested for its ability to lyse rabbit erythrocytes. 0.1% triton X was used as a positive control. Hemolysis was scored in percentage relative to the DMSO control set to 100%. Data are a representative of at least two independent, biological replicates.
Figure 7.
Solonamide B reduces psmα and agrA expression.
Strains USA300 or 8325-4 were grown exponentially to OD600 = 0.4 with either 5 µg/mL Solonamide B, DMSO or nothing added. RNA was purified from samples collected at OD600 = 0.7 and 1.7, and analyzed by Northern blotting. The membrane was probed with radioactive labeled probes targeting agrA and psmα.
Figure 8.
Solonamide B protects against S. aureus mediated neutrophil lysis.
Sterile filtered supernatants of S. aureus USA300 (S.a.) cultures grown for 7 hours or overnight (ON) with either Solonamide B at 5 µg/mL or 10 µg/mL or DMSO (control) were added to isolated human neutrophils. The control supernatant where only DMSO was added was tested in 1-fold, 3-fold and 9-fold dilutions (A). Undiluted (1∶1) supernatants from Solonamide B (SolB) treated cultures are shown together with the control in (B) and 3-fold dilutions (1∶3) are shown in (C). Lysis was monitored by lactate dehydrogenase (LDH) release. Data represents 3 independent experiments, using the average of triplicate wells from each experiment. Asteriks indicate SolB treated cultures resulting in lysis statistically significant from the corresponding control. *, p<0.05, **, p<0.01; ***, p<0.001.