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

Plasmids used in this study.

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

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

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</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

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