RsmA Regulates <i>Aspergillus fumigatus</i> Gliotoxin Cluster Metabolites Including Cyclo(L-Phe-L-Ser), a Potential New Diagnostic Marker for Invasive Aspergillosis

<div><p>Dimeric basic leucine zipper (bZIP) proteins are conserved transcriptional enhancers found in all eukaryotes. A recently reported and novel function for bZIPs is association of these proteins with secondary metabolite production in filamentous fungi. In particular a Yap-like bZIP termed RsmA (<u>r</u>estorer of <u>s</u>econdary <u>m</u>etabolism A) was identified in <i>Aspergillus nidulans</i> that positively regulates the carcinogen sterigmatocystin. To assess for conserved function for RsmA, we examined a role of this protein in secondary metabolism in the pathogen <i>A. fumigatus.</i> RsmA was found to positively regulate gliotoxin where overexpression (OE) of <i>rsmA</i> led to 2–100 fold increases of twelve <i>gli</i> cluster metabolites in culture medium including the newly identified <i>gli</i> metabolite cyclo(L-Phe-L-Ser). Lungs from both wild type and <i>OErsmA</i> infected mice contained gliotoxin (2.3 fold higher in <i>OErsmA</i> treatment) as well as the gliotoxin precursor cyclo(L-Phe-L-Ser) (3.2 fold higher in <i>OErsmA</i> treatment). The data here presents a conserved role for RsmA in secondary metabolite cluster activation and suggests cyclo(L-Phe-L-Ser) may serve as an alternative marker for diagnosis of invasive aspergillosis.</p></div

Infected mouse lung extracts (IMLE) HPLC- single ion monitoring (SIM)MS ion chromatograms.

<p>HPLC-SIMMS analysis of crude mouse lung extracts corresponding to mice infected with wild type, or <i>OErsmA</i>. (<b>A</b>) Ion chromatogram showing compound <b>6</b> is approximately two to three times as abundant in the <i>OErsmA</i>-IMLE relative to WT-IMLE. (<b>B</b>) Similarly, gliotoxin is about two times as abundant in <i>OErsmA</i>-IMLE than WT-IMLE. Reference chromatograms (bottom panels) show diagnostic ions of cyclo(L-Phe-L-Ser) (<b>6</b>) and gliotoxin (<b>1</b>). Lung extract chromatograms are scaled to the peaks measured in the <i>OErsmA</i>-IMLE sample (bottom panels of standards are not to scale).</p

Plasmids used in this study.

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

Oligonucleotides used in this study.

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

Neutrophil chemotaxis is reduced when exposed to extracts from <i>OErsmA</i>.

<p><b>A.</b> Schematic of the neutrophil migration platform used. The neutrophils are placed in the center channel; the compound to be tested is placed in one of the side channels, while the other channel acts as a negative control. The device is prepared in 3 steps: (1) filling, (2) adding neutrophils, and (3) adding the fungal culture supernatants. <b>B</b>. Representative microscopy images of the migration area after 1 h incubation. Supernatant of wild type <i>A. fumigatus</i> (AF WT) has chemoattractive properties on par with the known chemoattractant fMLP. <b>C</b>. Quantification of neutrophil recruitment to the crude supernatant of wild type, <i>OErsmA</i>, and <i>OErsmAΔgliZ</i> strains (significant differences at P<0.5 are indicated by different letters).</p

<i>OErsmA</i> mutants are resistant to menadione.

<p>For each strain, 10⁵ conidia in 5 µl were spotted on GMM plates with 20 µM, 30 µM and 40 µM of menadione, or on GMM only for a control. Each strain was replicated 5 times. The plates were incubated at 37°C for 48 h.</p

Average radial growth of <i>A. fumigatus</i> strains.

<p>10⁴ conidia of each strain were point inoculated on GMM and grown at 37°C for 4 days and at 25°C for 12 days (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062591#pone-0062591-g001" target="_blank">Figure 1</a>). Radial growth was measured at the end of each growth period. Means ± standard deviations are indicated for four replicates of each strain. Levels not connected by same letter are significantly different (<i>P</i><0.0001) according to ANOVA analysis.</p

Discovery of a Novel Pharmacological and Structural Class of Gamma Secretase Modulators Derived from the Extract of Actaea racemosa

A screen of a library of synthetic drugs and natural product extracts identified a botanical extract that modulates the processing of amyloid precursor protein (APP) in cultured cells to produce a lowered ratio of amyloid-beta peptide (1–42) (Aβ42) relative to Aβ40. This profile is of interest as a potential treatment for Alzheimer’s disease. The extract, from the black cohosh plant (Actaea racemosa), was subjected to bioassay guided fractionation to isolate active components. Using a combination of normal-phase and reverse-phase chromatography, a novel triterpene monoglycoside, <b>1</b>, was isolated. This compound was found to have an IC₅₀ of 100 nM for selectively reducing the production of amyloidogenic Aβ42 while having a much smaller effect on the production of Aβ40 (IC₅₀ 6.3 μM) in cultured cells overexpressing APP. Using IP-MS methods, this compound was found to modulate the pool of total Aβ produced by reducing the proportion of Aβ42 while increasing the relative amounts of shorter and less amyloidogenic Aβ37 and Aβ39. Concentrations of <b>1</b> sufficient to lower levels of Aβ42 substantially (up to 10 μM) did not significantly affect the processing of Notch or other aspects of APP processing. When <b>1</b> (10 μg) was administered to CD-1 normal mice intracerebroventricularly, the level of Aβ42 in brain was reduced. Assays for off-target pharmacology and the absence of overt signs of toxicity in mice dosed with compound <b>1</b> suggest a comparatively selective pharmacology for this triterpenoid. Compound <b>1</b> represents a new lead for the development of potential treatments for Alzheimer’s disease via modulation of gamma-secretase