Induction of Gliotoxin Secretion in Aspergillus fumigatus by Bacteria-Associated Molecules K. Stefan Svahn1, Ulf Go¨ ransson1, Erja Chryssanthou2, Bjo¨ rn Olsen3, Jan Sjo¨ lin3, Adam A. Stro¨ mstedt1* 1 Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden, 2 Department of Clinical Microbiology, Karolinska University Hospital & Karolinska Institute, Stockholm, Sweden, 3 Department of Medicinal Sciences, Uppsala University, Uppsala, Sweden Abstract Aspergillus fumigatus is the most common causative agent of mold diseases in humans, giving rise to life-threatening infections in immunocompromised individuals. One of its secreted metabolites is gliotoxin, a toxic antimicrobial agent. The aim of this study was to determine whether the presence of pathogen-associated molecular patterns in broth cultures of A. fumigatus could induce gliotoxin production. Gliotoxin levels were analyzed by ultra-performance liquid chromatography and mass spectrometry. The presence of a bacteria-derived lipopolysaccharide, peptidoglycan, or lipoteichoic acid in the growth media at a concentration of 5 mg/ml increased the gliotoxin concentration in the media by 37%, 65%, and 35%, respectively. The findings reveal a correlation between the concentrations of pathogen-associated molecular patterns and gliotoxin secretion. This shows that there is a yet uncharacterized detection system for such compounds within fungi. Inducing secondary metabolite production by such means in fungi is potentially relevant for drug discovery research. Our results also give a possible explanation for the increased virulence of A. fumigatus during bacterial co-infection, one that is important for the transition from colonization to invasiveness in this pulmonary disease. Citation: Svahn KS, Go¨ransson U, Chryssanthou E, Olsen B, Sjo¨lin J, et al. (2014) Induction of Gliotoxin Secretion in Aspergillus fumigatus by Bacteria-Associated Molecules. PLoS ONE 9(4): e93685. doi:10.1371/journal.pone.0093685 Editor: Gustavo Henrique Goldman, Universidade de Sao Paulo, Brazil Received October 31, 2013; Accepted March 9, 2014; Published April 4, 2014 Copyright: ß 2014 Svahn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been financially supported by the Swedish Governmental Agency for Innovation Systems - VINNOVA (http://www.vinnova.se/en). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction nents of the pathogens cytoplasmic barrier, which can act as general recognition targets for detecting their presence. The The discovery of penicillin based on observations made when detection of PAMPs by the immune system of insects and Penicillium rubens and Staphylococcus aureus were co-cultured on the mammals stimulates innate immune responses and thus prevents same agar plate was arguably the starting point of the Golden Era infections [6]. Some molecules that have been classified as PAMPs of Antibiotics [1,2]. However, P. rubens does not provide penicillin include lipopolysaccharides (LPS) from Gram-negative bacteria, in sufficient quantities for mass production under conventional lipoteichoic acid (LTA) from Gram-positive bacteria, and pepti- broth conditions. This problem was solved by the discovery of P. doglycan (PG) associated with both groups of bacteria. These chrysogenum, which has much higher levels of constitutive penicillin bacterial cell wall and membrane components induce responses in production. Laborious mutation and selection efforts eventually mammalian hosts by binding to Toll-like receptors (TLRs), which yielded a P. chrysogenum strain whose penicillin output exceeded are a subfamily of pattern recognition receptors (PRRs). TLRs that of Fleming’s mold by a factor of 1000 [3]. have been identified in a diverse range of animals but not in fungi To conserve nutrients and avoid self-toxicity as well as [7–10]. Although fungi seem to lack TLRs, it is important to know resistance development, organisms produce and secrete many if also fungi have some mechanism for detecting and responding to antimicrobial agents on a facultative basis. This is especially true common bacterial PAMPs. for microorganisms such as fungi, which cannot rely as much on The fungus A. fumigatus is the most common causative agent of nutrient depots and cell-specialization. As a result, fungi would mold infections in humans [11]. Pathogenic species of the genus benefit especially from having metabolic pathways that are Aspergillus often infect severely immunocompromised patients as dormant or downregulated where there are no competing microorganisms. Consequently, it is disputable that fungi secrete well as those with more moderate levels of immunosuppression, the full spectrum of antimicrobial agents in conventional such as patients with chronic obstructive pulmonary disease laboratory growth conditions. Methods for activating these (COPD) or critically ill intensive care patients, in whom it can pathways could therefore be useful for finding new metabolites cause serious invasive infections with high mortality rates [11]. or increasing the secretion of known ones, which may be beneficial The aim of the work reported herein was to determine how in drug discovery and production. three PAMPs (LPS, LTA and PG) affect the secreted metabolite Pathogen-associated molecular patterns (PAMPs) are widely profile of A. fumigatus. The use of bacteria or PAMPs to induce used to simulate the presence of bacteria and other pathogens in fungal metabolite production represents a novel methodology. The studies on mammalian immune systems [4], [5]. PAMPs are traditional way of exploring fungal secondary metabolites is to highly conserved molecular structures, typically essential compo- cultivate them in a standard medium and then analyze the culture PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e93685 Bacterial Motifs Induce Gliotoxin in Aspergillus filtrate or fungal mycelium [12]. Elicitation using PAMPs could be After seven days of incubation, the culture broths from the pilot particularly useful because it is likely that many bioactive fungal study were analyzed qualitatively using high performance liquid metabolites are produced facultatively and so would not be chromatography (HPLC), ultra performance liquid chromatogra- observed under standard cultivation conditions. It was expected phy (UPLC), both reverse phase, and mass spectrometry (MS). that the results obtained in this way might improve our The HPLC-derived metabolite profiles of the PAMP-treated understanding of the pathogenesis of fungal infection, especially cultures showed a marked increase in the gliotoxin peak whiles the transition from colonization to invasion, and the problems otherwise strikingly similar to those of the controls (Figure 2). The associated with bacterial co-infections [13]. A. fumigatus was culture filtrates and mycelial extracts were analyzed in an selected as the object of study because of its potential to provide antimicrobial assay to determine whether they contained any clinically relevant information as well as new insights into the metabolites with antimicrobial activity. The only HPLC fractions effects of PAMPs on fungal metabolism. of the filtered broths that exhibited antimicrobial activity were those that contained gliotoxin. These gliotoxin-containing frac- Results tions inhibited bacterial growth down to a maximum dilution of 1:64. None of the solid phase extracts of the fungal mycelia showed A. fumigatus cultures were grown in the presence of the three any antimicrobial activity, and had similar MS-profiles (data not selected PAMPs at various concentrations for seven days and the shown). The near-identical metabolite profiles of the treated and gliotoxin production in each case was compared to that of untreated cultures and the lack of antimicrobial activity in the untreated control cultures. The minimum PAMP concentrations gliotoxin-free HPLC fractions of the broths and mycelial extracts required to cause appreciable increases in gliotoxin secretion were indicate that A. fumigatus does not produce additional antimicrobial 0.3 mg PG/ml, 1 mg LPS/ml, and 5 mg LTA/ml (Figure 1). The secondary metabolites in the presence of these PAMPs. level of gliotoxin release into the media increased with the level of Over a 13-day period (Figure 3), the differences between the PAMP concentration, up to 10 mg/ml of each PAMP beyond gliotoxin levels produced by PAMP-treated A. fumigatus cultures which the gliotoxin increase subsided. and untreated controls were comparable to those observed in the To measure the filtration efficiency, ten 1 ml samples of 1 mM preliminary 7-day experiments. The gliotoxin concentration was gliotoxin were prepared. Half of the volume of each sample was highest between days five and eleven, and tended to increase more repeatedly filtered ten times and compared with the unfiltered part rapidly and to peak at a higher level in cultures that were exposed of the sample. The efficiency was higher than 99.5% per filtration. to PAMPs. The addition of LPS, LTA and PG at a concentration Cultures supplemented with all three PAMPs at a concentration of 5 mg per ml of culture