The authors have declared that no competing interests exist.
Current address: Novo Nordisk, Gentofte, Denmark
Current address: Agilent Technologies, Glostrup, Denmark
Current address: Gentofte Hospital, Hellerup, Denmark
Several natural compounds have been found to target virulence gene expression in
For RNA isolation cells were inoculated at OD600 = 0.02–0.03. Norlichexanthone was added at OD600 = 0.4. Samples for RNA purification were taken at OD600 = 0.7 and/or 2.0 for strain USA300 and strain Newman, and 1 hour after addition of norlichexanthone for strain 8325–4. Cells were harvested and total RNA was extracted using RNeasy kit (Qiagen) in accordance with the manufacturer’s instructions. RNA quantity and quality were measured on a NanoDrop ND-1000 spectrophotometer by absorbance (A260, A260/A280 and A260/A230 ratio respectively) and by gel electrophoresis. Northern blot analysis was performed as described previously using equal amounts of total RNA for each lane [
Gene | Forward primer | Reverse primer |
---|---|---|
RNAIII | ||
Northern blot primers | ||
RNAIII | ||
The ability of
The assay was performed as described [
P3 promoter activity was measured in strain RN10829 expressing in trans either wild type AgrC (p
The C-terminal, DNA binding domain of AgrA (AgrAc) from 8325–4 was PCR amplified (using the AgrAc forward and reverse primers) and cloned between the
The P2-P3 probe (49 bp duplex DNA) covering the
Previously we used transcriptional reporter gene fusions to show that norlichexanthone extracted from
Expression of
Structurally, norlichexanthone is almost identical to ω-hydroxyemodin and bears some resemblance to savirin (
(A) Activity of the P3
To experimentally address the possibility that norlichexanthone interferes with AgrA binding to DNA, we expressed the C-terminal DNA binding domain of AgrA (AgrAc) and examined its effect on AgrAc binding to the P2-P3 probe carrying AgrA binding sites using the electrophoretic mobility shift assay (EMSA) (
Samples including AgrAc, DNA probe, and/or compound were loaded in TBE buffer containing 10 mM dithiothreitol. Assays including the P2-P3 49 bp probe were analyzed in 4.5% native polyacrylamide gels. Lanes: 1. Size marker; 2. P2-P3 probe alone; 3. Probe and norlichexanthone (Nor) in the absence of AgrAc protein; 4. 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.
Human neutrophils are one of the primary defences against
Sterile filtered supernatants of
The success of
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°C. (A) Quantification of aggregation. After sedimentation, the supernatants of the cultures were measured for optical density at 600 nm. (B) Dilutions were inoculated into a 96-well microtiter plate containing 200 μL TSB. Norlichexanthone was added to a final concentration of 0.5, 1, 5 and 10 μg/mL. No inoculate, no treatment and DMSO (compound vehicle) were used as controls. The plate was incubated for 20 hours at 37°C without shaking. Biofilm formation was quantified by 0.1% crystal violet staining and OD measurement at 590 nm. (C) Culture CFUs were obtained to eliminate possibility of compound toxicity attributing to reduction of aggregation. Each bar represents the average of 3 replicates and the error bars represent the standard deviation.
In an effort to better understand the effect of norlichexanthone on
Interestingly, among the genes affected by norlichexanthone is a group that is regulated by the two component transcriptional regulatory system, SaeRS namely
To further implicate the SaeRS system in response to norlichexanthone we examined the effect of the compound in strain Newman that naturally harbors a mutation in the
Expression was monitored in strains USA300 (FPR3757) and Newman by quantitative RT-PCR. Depicted are fold-changes of the transcripts observed upon treatment with 5 μg/ml norlichexanthone relative to DMSO from samples taken at OD600 = 2.0. The data represent the mean and standard deviation from 3 biological replicates.
Our results show that norlichexanthone reduces virulence gene expression in the CA-MRSA strain USA300 and that it modulates virulence gene expression by interfering with at least two different key virulence gene regulons; the
Strain 8325–4 was grown exponentially to OD600 = 0.4 where either 5 μg/mL norlichexanthone or DMSO was added. Transcript levels after 1 hour treatment were determined by RT-qPCR. Depicted mean fold-changes (+/- standard deviation) by norlichexanthone are based upon 3 biological replicates.
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Strain USA300 were grown exponentially to OD600 = 0.4 where either 5 μg/mL norlichexanthone (nor), DMSO or nothing was added. RNA was purified from samples collected at OD600 = 0.7 and 2.0, and analyzed by Northern blotting where equal amounts of RNA was loaded. The membrane was probed with radioactive labeled probes targeting
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Cells were grown in TSB and treated with 5 μg/mL norlichexanthone or corresponding amount of DMSO at OD600 = 0.4. Data is based on biological triplicates, sampled at OD600 = 2.0. Fold regulation indicates norlichexanthone treated cells relative to DMSO treated cells.
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Christiane Wolz and Alex Horswill kindly provided constructive comments to the results.