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Genetic and Structural Validation of Phosphomannomutase As a Cell University of Dundee Genetic and structural validation of phosphomannomutase as a cell wall target in Aspergillus fumigatus Zhang, Yuanwei; Fang, Wenxia; Raimi, Olawale G.; Lockhart, Deborah; Ferenbach, Andrew T.; Lu, Ling Published in: Molecular Microbiology DOI: 10.1111/mmi.14706 Publication date: 2021 Licence: CC BY Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal Citation for published version (APA): Zhang, Y., Fang, W., Raimi, O. G., Lockhart, D., Ferenbach, A. T., Lu, L., & van Aalten, D. (2021). Genetic and structural validation of phosphomannomutase as a cell wall target in Aspergillus fumigatus. Molecular Microbiology, 116(1), 245-259. https://doi.org/10.1111/mmi.14706 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 25. Sep. 2021 Received: 5 October 2020 | Revised: 3 February 2021 | Accepted: 15 February 2021 DOI: 10.1111/mmi.14706 RESEARCH ARTICLE Genetic and structural validation of phosphomannomutase as a cell wall target in Aspergillus fumigatus Yuanwei Zhang 1,2 | Wenxia Fang2,3 | Olawale G. Raimi2 | Deborah E. A. Lockhart2 | Andrew T. Ferenbach2 | Ling Lu1 | Daan M. F. van Aalten 2 1Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering Abstract and Technology Research Centre for Aspergillus fumigatus is an opportunistic mold responsible for severe life- threatening Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China fungal infections in immunocompromised patients. The cell wall, an essential 2School of Life Sciences, University of structure composed of glucan, chitin, and galactomannan, is considered to be a Dundee, Dundee, UK target for the development of antifungal drugs. The nucleotide sugar donor GDP- 3National Engineering Research Center for Non- Food Biorefinery, Guangxi Academy of mannose (GDP- Man) is required for the biosynthesis of galactomannan, glyco- Sciences, Nanning, China sylphosphatidylinositol (GPI) anchors, glycolipid, and protein glycosylation. Starting Correspondence from fructose- 6- phosphate, GDP- Man is produced by the sequential action of the Daan M. F. van Aalten, School of Life enzymes phosphomannose isomerase, phosphomannomutase (Pmm), and GDP- Sciences, University of Dundee, Dundee DD1 5EH, UK. mannose pyrophosphorylase. Here, using heterokaryon rescue and gene knockdown Email: [email protected] approaches we demonstrate that the phosphomannomutase encoding gene in A. fu- Funding information migatus (pmmA) is essential for survival. Reduced expression of pmmA is associated Medical Research Council, Grant/Award with significant morphological defects including retarded germination, growth, re- Number: V001094 duced conidiation, and abnormal polarity. Moreover, the knockdown strain exhibited an altered cell wall organization and sensitivity toward cell wall perturbing agents. By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non- conservative substitutions near the active site when compared to the human orthologues. Taken together, this work provides a genetic and structural foundation for the exploitation of AfPmmA as a potential antifungal target. KEYWORDS Aspergillus fumigatus, crystal structure, drug target, phosphomannomutase 1 | INTRODUCTION Robbins et al., 2017). Only four structural classes of antifungal drugs (azoles, polyenes, echinocandins, and flucytosine) are used Aspergillus fumigatus is a ubiquitous and opportunistic filamentous in the clinical treatment of fungal infections (Ostrosky- Zeichner fungus that causes life- threatening invasive aspergillosis with a high et al., 2010) and no novel antifungal classes have been discovered mortality rate in immunocompromised individuals such as bone mar- since 2006 (Denning & Bromley, 2015). Clinical development of row and solid organ transplant recipients (Brown et al., 2012; Kousha novel therapeutics is mainly limited to combinations of existing et al., 2011). The therapeutic options for A. fumigatus infections are drugs, repurposing medications or novel synthetic molecules with restricted due to limited antifungal repertoire, severe side effects in unknown mechanisms of action (Calderone et al., 2014). Thus, these patients and emergence of drug resistance (Bongomin et al., 2017; problems highlight the importance of identifying and characterizing This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd. Molecular Microbiology. 2021;00:1–15. wileyonlinelibrary.com/journal/mmi | 1 2 | ZHANG ET AL. new antifungal targets against A. fumigatus in order to feed into drug discovery (FBDD) (Erlanson et al., 2016), it is possible to identify discovery pipelines. weakly binding small fragments (usually molecular mass < 300 Da) The fungal cell wall is a complex and highly dynamic structure targeting binding sites away from highly conserved active sites. that is essential for cellular morphology and protection against These fragments can be converted to potent inhibitors with high environmental stresses and is considered to be a potential drug selectivity by iterative optimization based on structural and en- target. It is primarily composed of the polysaccharides chitin, glu- zymological information (Scott et al., 2012). To date, although can, and galactomannan (Gow et al., 2017; Latge et al., 2005). In A. the function of phosphomannomutase has been well character- fumigatus, galactomannan is a highly complex structure containing ized in many eukaryotes, the physiological function of PmmA in different types of glycosidic linkages, produced by several manno- the human opportunistic pathogen A. fumigatus remains unclear. syltransferases (Lee & Sheppard, 2016). As an active form of man- Moreover, active site sequence conservation of this enzyme fam- nose, GDP- mannose is not only required for biosynthesis of fungal ily with the human orthologues hampers the development of galactomannan, but also plays an important role in biosynthesis of specific inhibitors. Here, we demonstrate that AfPmmA is indis- O- and N- linked glycoproteins, glycosylphosphatidylinositol (GPI) pensable for viability, morphogenesis, and cell wall integrity in A. anchors and glycolipids (Latge et al., 2017; Onoue et al., 2018). fumigatus. Importantly, we reveal potential exploitable differences GDP- mannose biosynthesis is catalyzed by a cascade of three en- in AfPmmA structure compared to its human orthologues. This zymes, starting from fructose 6- phosphate (Fru- 6P), converted to work forms the basis for the initiation of structure- based inhibitor mannose 6- phosphate (Man- 6P) by mannose 6- phosphate isom- design against AfPmmA. erase (Pmi), isomerized to mannose 1- phosphate by phospho- mannomutase (Pmm), and finally converted to GDP- mannose in the presence of GTP by GDP- mannose pyrophosphorylase (Gmp) 2 | RESULTS (Sharma et al., 2014). Pmm, the second enzyme in GDP- mannose biosynthesis, belongs to the haloalkanoic acid dehalogenase 2.1 | A. fumigatus possesses a functional (HAD) superfamily, containing a conserved phosphorylated motif phosphomannomutase DxDx(T/V) and utilizing a bisphosphate sugar (either glucose 1,6- bisphosphate or mannose 1,6- bisphosphate) as a co- factor Using the Genbank accession codes of H. sapiens Pmm1 (Allen & Dunaway- Mariano, 2004; Collet et al., 1998). Published (NP_002667.2) and Pmm2 (NP_000294.1) for a BLASTp search crystal structures of Pmm from Homo sapiens, Leishmania mexicana, in A. fumigatus A1163 genome yielded a single putative phos- and Candida albicans revealed conserved overall structures with a phomannomutase (AfPmmA, EDP49225.1) corresponding to the similar catalytic mechanism of this enzyme family (Ji et al., 2018; pmmA gene (AFUB_072510). A. fumigatus pmmA is located on Kedzierski et al., 2006; Silvaggi et al., 2006). Since GDP- mannose chromosome 6 and 1,087 bp in length containing four exons and is a key metabolic intermediate for many cellular processes, Pmms three introns. The encoded protein AfPmmA contains 245 amino have been identified and genetically characterized in eukaryotes. acids with 49% and 53% identity to human Pmm1 and Pmm2, For example, Saccharomyces cerevisiae Pmm was shown to be es- respectively. AfPmmA is predicted to catalyze the conversion sential for cell viability under in vitro laboratory conditions (Kepes of mannose- 6- phosphate (Man- 6P) to mannose- 1- phosphate & Schekman, 1988). Similar findings were observed in Arabidopsis (Man- 1P) in the synthesis pathway of GDP– mannose (GDP- Man), thaliana, where failure of obtaining
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