Prototype of an intertwined secondary-metabolite supercluster

Abstract

The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the control of the master regulator of secondary metabolism, LaeA, contains, in its entirety, the genetic machinery for three natural products (fumitremorgin, fumagillin, and pseurotin), where genes for fumagillin and pseurotin are physically intertwined in a single supercluster. Deletions of 29 adjoining genes revealed that fumagillin and pseurotin are coregulated by the supercluster-embedded regulatory gene with biosynthetic genes belonging to one of the two metabolic pathways in a noncontiguous manner. Comparative genomics indicates the fumagillin/pseurotin supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This blended design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution.gene regulation | Zn(II) 2 Cys 6 transcription factor | FapR | biosynthesis | cluster evolution F ilamentous fungi are well known for their ability to produce a variety of natural products, so-called secondary metabolites that are not essential for growth under laboratory conditions (reviewed in refs. 1 and 2). However, the maintenance of the genetic information allowing fungi to produce secondary metabolites suggests that these small molecules provide essential benefits in environmental niches ranging from protection from fungivory (reviewed in ref.3) to chemical shields from UV radiation (4). Apart from providing evolutionary fitness to the producing organism in their natural habitat, many secondary metabolites are of major importance to humans because of their beneficial and deleterious effects as drugs and toxins, respectively.Fungal secondary metabolism has been characterized by physical linkage of the genes required for synthesis of specific metabolites and the distinct enzymatic machinery encoded by these genes (1, 2, 5). For instance, most fungal secondary metabolites belong to one of four chemical classes that are characterized based on the key or backbone enzymes that consist of polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), terpene cyclases (TCs), and prenyltransferases (PTs). Typically, cluster genes adjacent to these backbone genes code for accessory enzymes involved in either modification of the chemical scaffold, transcriptional control of cluster genes, transport of substrates and/or products, and resistance mechanisms. The most common regulatory genes of clusters encode fungal-specific C6 zinc binuclear cluster (Zn(II) 2 Cys 6 ) transcription factors (6), which, in general, exert positive transcriptional regulation of most of the genes within a single cluster (7). In addition to cluster-specific transcription factors, a higher order...

A possible role for fumagillin in cellular damage during host infection by Aspergillus fumigatus

“…These included the fumagillin/pseurotin transcription factor FapR [24] (also known as FumR [26]), four fumagillin biosynthetic genes, one of the two non-canonical methionine aminopeptidase encoding genes located inside the supercluster region, implicated to convey fumagillin resistance ( fpaII ; Afu8g00410), five pseurotin biosynthetic genes and three genes of unknown function. Additional DEGs included genes codifying for hydrolytic enzymes such as DppIV (Afu4g09320), DppV (Afu2g00930), the allergens Asp f 1 (Afu5g02330) and Asp f 5 or elastinolytic metalloproteinase Mep (Afu8g07080) and the chitinase ChiB1 (Afu8g01410), among others (Table S2).…”

“…To address a possible role of either metabolite in host cell damage, we analyzed the interactions with the A549 pulmonary epithelial cell line of wild type A. fumigatus , a fumagillin mutant strain, ∆fmaA , which still produces pseurotin, and a pseurotin mutant strain, ∆psoA , which still produces fumagillin [24]. …”

“…Among them, the 14 (out of 21) genes belonging to the intertwined fumagillin/pseurotin biosynthetic cluster were notable, including the essential transcription factor FumR/FapR (Afu8g00420) critical for the production of both metabolites [24,26]. Our transcriptome data showed that fapR expression was early with increases of expression of the fma/pso biosynthetic genes with invasion time.…”

“…After harvesting and cleaning twice with a saline-Tween solution (0.9% NaCl, 0.02% Tween 20) (SS-Tw20), the number of conidia was calculated using a haemocytometer and their viability tested by plating them onto potato dextrose agar (Cultimed) and expressed as a percentage. Furthermore, the previously published [24] deletion mutants strains ∆fmaA (fumagillin − pseurotin + ) and ∆psoA (fumagillin + pseurotin − ) were used for the fumagillin 51 Cr release assays and HPLC detection assays, alongside their correspondent wild type strain A. fumigatus CEA17.…”

“…Transcriptomic profiles from early (24 h) and advanced (72 and 96 h) stages of infection were compared to identify gene expression patterns, which were verified by RT-qPCR. Notably, 14 genes of the 21 intertwined fumagillin/pseurotin gene cluster [24] were found to be upregulated at the advanced time points. Furthermore, an A. fumigatus ∆fmaA (fumagillin null) mutant caused significantly less damage that the wild-type strain to a pulmonary epithelial cell line in vitro , suggesting a role for fumagillin in inducing cellular injury.…”