Imaging mass spectrometry-guided fast identification of antifungal secondary metabolites from Penicillium polonicum
The discovery of antibiotics from microorganisms using classic bioactivity screens suffers from heavy labor and high re-discovery rate. Recently, largely uncovered biosynthetic potentials were unveiled by new approaches, such as genetic manipulation of “silent” biosynthetic gene clusters, innovative data acquisition, and processing methods. In this work, a fast and efficient antibiotic identification pipeline based on the MALDI-TOF imaging mass spectrometry was applied to study the antifungal metabolites during the confrontation of two fungal species, Penicillium polonicum and wilt-inducing fungus Fusarium oxysporum. By visualizing the spatial distribution of metabolites directly on the microbial colony and surrounding media, we predicted the antifungal candidates before isolating pure compounds and individually testing their bioactivity, which subsequently guided the identification of target molecules using classic chromatographic methods. Via this procedure, we successfully identified two antifungal metabolites, fructigenine A and B, which belong to indole alkaloid class and were not reported for antifungal activity. Our work assigned new bioactivity to previously reported compounds and more importantly showed the efficiency of this approach towards quick discovery of bioactive compounds, which can help study the vast unexploited synthetic potential of microbial secondary metabolites.
KeywordsMALDI-TOF imaging mass spectrometry Penicillium polonicum Antifungal secondary metabolite Indole alkaloid
We thank Agricultural Culture Collection of China (ACCC) and Professor Bingyan Xie from Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, for generously providing the Penicillium polonicum strains. We also thank the Research Facility Center of the Biotechnology Institute for providing equipment, HPLC-HRMS (Agilent, USA), and the software.
This work was supported by the “948” Project of the Ministry of Agriculture of China (2016-X43 to Y.X.), National Natural Science Foundation of China (31500079 to LW.Z. and 31570093 to Y.X.), The Agricultural Science and Technology Innovation Program (CAAS-XTCX2016012 to Y.X.), and the key project of the China National Tobacco Corporation (110201502019 to Y.X.).
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare no conflict of interest, and that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Chai Y-J, Cui C-B, Li C-W, Wu C-J, Tian C-K, Hua W (2012) Activation of the dormant secondary metabolite production by introducing gentamicin-resistance in a marine-eerived Penicillium purpurogenum G59. Mar Drugs 10(3):559–582. https://doi.org/10.3390/md10030559 CrossRefPubMedPubMedCentralGoogle Scholar
- Clevenger KD, Bok JW, Ye R, Miley GP, Verdan MH, Velk T, Chen C, Yang K, Robey MT, Gao P, Lamprecht M, Thomas PM, Islam MN, Palmer JM, Wu CC, Keller NP, Kelleher NL (2017) A scalable platform to identify fungal secondary metabolites and their gene clusters. Nat Chem Biol 13(8):895–901. https://doi.org/10.1038/nchembio.2408 CrossRefPubMedPubMedCentralGoogle Scholar
- Frisvad JC, Smedsgaard J, Larsen TO, Samson RA, Robert A (2004) Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Stud Mycol 49(201):e41Google Scholar
- Gonzalez DJ, Xu Y, Yang YL, Esquenazi E, Liu WT, Edlund A, Duong T, Du L, Molnar I, Gerwick WH, Jensen PR, Fischbach M, Liaw CC, Straight P, Nizet V, Dorrestein PC (2012) Observing the invisible through imaging mass spectrometry, a window into the metabolic exchange patterns of microbes. J Proteome 75(16):5069–5076. https://doi.org/10.1016/j.jprot.2012.05.036 CrossRefGoogle Scholar
- Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD (2017) A link between linearmycin biosynthesis and extracellular vesicle genesis connects specialized metabolism and bacterial membrane physiology. Cell chem Biol 24(10):1238–1249.e7. https://doi.org/10.1016/j.chembiol.2017.08.008 CrossRefPubMedGoogle Scholar
- Kaur T, Kaur A, Sharma V, Manhas RK (2016) Purification and characterization of a new antifungal compound 10-(2,2-dimethyl-cyclohexyl)-6,9-dihydroxy-4,9-dimethyl-dec-2-enoic acid methyl ester from Streptomyces hydrogenans strain DH16. Front Microbiol 7:1004. https://doi.org/10.3389/fmicb.2016.01004 CrossRefPubMedPubMedCentralGoogle Scholar
- Kersten RD, Yang YL, Xu Y, Cimermancic P, Nam SJ, Fenical W, Fischbach MA, Moore BS, Dorrestein PC (2011) A mass spectrometry-guided genome mining approach for natural product peptidogenomics. Nat Chem Biol 7(11):794–802. https://doi.org/10.1038/nchembio.684 CrossRefPubMedPubMedCentralGoogle Scholar
- Michelsen CF, Watrous J, Glaring MA, Kersten R, Koyama N, Dorrestein PC, Stougaard P (2015) Nonribosomal peptides, key biocontrol components for Pseudomonas fluorescens In5, isolated from a greenlandic suppressive soil. Mbio 6(2):e00079–e00015. https://doi.org/10.1128/mBio.00079-15 CrossRefPubMedPubMedCentralGoogle Scholar
- Nielsen JC, Grijseels S, Prigent S, Ji B, Dainat J, Nielsen KF, Frisvad JC, Workman M, Nielsen J (2017) Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species. Nat Microbiol 2:17044. https://doi.org/10.1038/nmicrobiol.2017.44 CrossRefPubMedGoogle Scholar
- Seneviratne HK, Dalisay DS, Kim KW, Moinuddin SGA, Yang H, Hartshorn CM, Davin LB, Lewis NG (2015) Non-host disease resistance response in pea (Pisum sativum) pods: biochemical function of DRR206 and phytoalexin pathway localization. Phytochemistry 113:140–148. https://doi.org/10.1016/j.phytochem.2014.10.013 CrossRefPubMedGoogle Scholar
- Wayne, PA (2002) National committee for clinical laboratory standards. Performance standards for antimicrobial disc susceptibility testing. 12:01–53Google Scholar
- Xin ZH, Zhu WM, Gu QQ, Fang YC, Duan L, Cui CB (2005) A new cytotoxic compound from Penicillium auratiogriseum, symbiotic or epiphytic fungus of sponge Mycale plumose. Chin Chem Lett 16(9):1227–1229Google Scholar
- Xin Z, Fang Y, Zhu T, Duan L, Gu Q, Zhu W (2006) Antitumor components from sponge-derived fungus Penicillium auratiogriseum Sp-19. Chin J Mar Drugs 25(6):1–6Google Scholar
- Yaegashi J, Oakley BR, Wang CCC (2014) Recent advances in genome mining of secondary metabolite biosynthetic gene clusters and the development of heterologous expression systems in Aspergillus nidulans. J Ind Microbiol Biotechnol 41(2):433–442. https://doi.org/10.1007/s10295-013-1386-z CrossRefPubMedGoogle Scholar
- Yang YL, Xu Y, Kersten RD, Liu WT, Meehan MJ, Moore BS, Bandeira N, Dorrestein PC (2011) Connecting chemotypes and phenotypes of cultured marine microbial assemblages by imaging mass spectrometry. Angew Chem Int Ed Engl 50(26):5839–5842. https://doi.org/10.1002/anie.201101225 CrossRefPubMedPubMedCentralGoogle Scholar