![]() Each bar represents the average of 3 replicates and the error bars represent the standard deviation.Įxpression of coa and hla monitored by quantitative RT-PCR.Įxpression was monitored in strains USA300 (FPR3757) and Newman by quantitative RT-PCR. (C) Culture CFUs were obtained to eliminate possibility of compound toxicity attributing to reduction of aggregation. Biofilm formation was quantified by 0.1% crystal violet staining and OD measurement at 590 nm. The plate was incubated for 20 hours at 37☌ without shaking. No inoculate, no treatment and DMSO (compound vehicle) were used as controls. Norlichexanthone was added to a final concentration of 0.5, 1, 5 and 10 μg/mL. (B) Dilutions were inoculated into a 96-well microtiter plate containing 200 μL TSB. After sedimentation, the supernatants of the cultures were measured for optical density at 600 nm. Strain 8325–4 was cultivated in the presence of DMSO, 0.5, 1, 5 μg/mL or 10 μg/mL norlichexanthone, and incubated 8½ hours at 37☌. *, p<0.05, **, p<0.01 ***, p<0.001.Īggregation and biofilm formation are significantly reduced by norlichexanthone. Asterisk indicates norlichexanthone treated cultures resulting in lysis statistically significant from the corresponding control by one-way ANOVA with Dunnett’s posttest. Data represents 3 independent experiments, using the average of triplicate wells from each experiment. Lysis was monitored by lactate dehydrogenase (LDH) release. Undiluted (1:1) supernatants from norlichexanthone (norlic) treated cultures are shown together with untreated supernatant in (B) and 3-fold dilutions (1:3) are shown in (C). The control supernatant where only DMSO was added was examined in 1-fold, 3-fold and 9-fold dilutions (A). aureus USA300 (S.a.) cultures grown for 7 hours with either norlichexanthone at 5 μg/mL or 10 μg/mL or DMSO (control) were added to isolated human neutrophils. 100 uM I-d (potential hit compound and positive binding control) 5–10: norlichexanthone (Nor) increased by two-fold concentrations from 0–100 μg/ml. Probe and norlichexanthone (Nor) in the absence of AgrAc protein 4. Assays including the P2-P3 49 bp probe were analyzed in 4.5% native polyacrylamide gels. Samples including AgrAc, DNA probe, and/or compound were loaded in TBE buffer containing 10 mM dithiothreitol. Norlichexanthone interferes with AgrA binding to DNA. ![]() The data represent the mean and standard deviation from 3 biological replicates with the effect of norlichexanthone being statistically significant ( P<0.01 at both time points, two-tailed student’s t-test). The β-lactamase activity was measured at 30 and 60 minutes and subtracted the BlaZ activity present already at time 0 to infer the effect on P3 expression after compound addition. (B) Accumulated activity of the P3 -blaZ promoter in a strain expressing a constitutively active AgrC receptor in presence or absence of 5 μg/ml norlichexanthone (Nor). In all conditions, the effect of norlichexanthone is statistically significant ( P<0.001, two-tailed student’s t-test). The data represent the mean and standard deviation of the β-lactamase activity obtained from 3 biological replicates at different AIP concentrations (5, 13, and 20%) at time points 30 and 60 minutes. ![]() (A) Activity of the P3 -blaZ promoter in a strain expressing a wild type, AIP-inducible AgrC receptor in presence or absence of 5 μg/ml norlichexanthone (Nor). Norlichexanthone reduces RNAIII expression in an AIP concentration-independent manner and also in a mutant with a constitutively active AgrC receptor. 1: no treatment, 2: DMSO, 3: Norlichexanthone 5 μg/mL, 4: no treatment, 5: DMSO, 6: Norlichexanthone 5 μg/mL. RNA was purified from samples taken at OD600 = 0.7 (lanes 1–3) and 2.0 (lanes 4–6). B) Norlichexanthone 5 μg/mL or DMSO or nothing was added at OD600 = 0.4. Norlichexanthone affects expression of spa, hla and RNAIII.Įxpression of hla, spa and RNAIII was examined by Northern blot analysis with probes recognizing the transcripts in strain 8325–4 (A) or USA300 (B) treated with in A): 1: No treatment, 2: DMSO (negative control), 3: Norlichexanthone 50 μg/mL (dissolved in DMSO), 4: Norlichexanthone 5 μg/mL, 5: Norlichexanthone 0.5 μg/mL, 6: Norlichexanthone 0.05 μg/mL, 7: Norlichexanthone 0.005 μg/mL added at OD600 = 0.4, and sampled after 1 hour. ![]()
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