Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

Staphylococcus aureus is a serious human pathogen and antibiotic resistant, community-associated strains, such as the methicillin resistant S. aureus (MRSA) strain USA300, continue to spread. To avoid resistance, anti-virulence therapy has been proposed where toxicity is targeted rather than viability. Previously we have shown that norlichexanthone, a small non-reduced tricyclic polyketide produced by fungi and lichens, reduces expression of hla encoding α-hemolysin as well as the regulatory RNAIII of the agr quorum sensing system in S. aureus 8325-4. The aim of the present study was to further characterise the mode of action of norlichexanthone and its effect on biofilm formation. We find that norlichexanthone reduces expression of both hla and RNAIII also in strain USA300. Structurally, norlichexanthone resembles ω-hydroxyemodin that recently was shown to bind the agr two component response regulator, AgrA, which controls expression of RNAIII and the phenol soluble modulins responsible for human neutrophil killing. We show that norlichexanthone reduces S. aureus toxicity towards human neutrophils and interferes directly with AgrA binding to its DNA target. In contrast to ω-hydroxyemodin however, norlichexanthone reduces staphylococcal biofilm formation. Transcriptomic analysis revealed that genes regulated by the SaeRS two-component system are repressed by norlichexanthone when compared to untreated cells, an effect that was mitigated in strain Newman carrying a partially constitutive SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation

Norlichexanthone affects expression of <i>spa</i>, <i>hla</i> and RNAIII.

<p>Expression of <i>hla</i>, <i>spa</i> 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. B) Norlichexanthone 5 μg/mL or DMSO or nothing was added at OD600 = 0.4. RNA was purified from samples taken at OD600 = 0.7 (lanes 1–3) and 2.0 (lanes 4–6). 1: no treatment, 2: DMSO, 3: Norlichexanthone 5 μg/mL, 4: no treatment, 5: DMSO, 6: Norlichexanthone 5 μg/mL.</p

Norlichexanthone interferes with AgrA binding to DNA.

<p>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.</p

Expression of <i>coa</i> and <i>hla</i> monitored by quantitative RT-PCR.

<p>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.</p

Norlichexanthone reduces RNAIII expression in an AIP concentration-independent manner and also in a mutant with a constitutively active AgrC receptor.

<p>(A) Activity of the P3<i>-blaZ</i> promoter in a strain expressing a wild type, AIP-inducible AgrC receptor in presence or absence of 5 μg/ml norlichexanthone (Nor). 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. In all conditions, the effect of norlichexanthone is statistically significant (<i>P</i><0.001, two-tailed student’s t-test). (B) Accumulated activity of the P3<i>-blaZ</i> promoter in a strain expressing a constitutively active AgrC receptor in presence or absence of 5 μg/ml norlichexanthone (Nor). 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. The data represent the mean and standard deviation from 3 biological replicates with the effect of norlichexanthone being statistically significant (<i>P</i><0.01 at both time points, two-tailed student’s t-test).</p

Primers used in this study.

<p>Primers used in this study.</p

Norlichexanthone protects against <i>S</i>. <i>aureus</i> mediated neutrophil lysis.

<p>Sterile filtered supernatants of <i>S</i>. <i>aureus</i> 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. The control supernatant where only DMSO was added was examined in 1-fold, 3-fold and 9-fold dilutions (A). 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). Lysis was monitored by lactate dehydrogenase (LDH) release. Data represents 3 independent experiments, using the average of triplicate wells from each experiment. Asterisk indicates norlichexanthone treated cultures resulting in lysis statistically significant from the corresponding control by one-way ANOVA with Dunnett’s posttest. *, p<0.05, **, p<0.01; ***, p<0.001.</p