Nancy P. Keller

Fungal secondary metabolism — from biochemistry to genomics

Much of natural product chemistry concerns a group of compounds known as secondary metabolites. These low-molecular-weight metabolites often have potent physiological activities. Digitalis, morphine and quinine are plant secondary metabolites, whereas penicillin, cephalosporin, ergotrate and the statins are equally well known fungal secondary metabolites. Although chemically diverse, all secondary metabolites are produced by a few common biosynthetic pathways, often in conjunction with morphological development. Recent advances in molecular biology, bioinformatics and comparative genomics have revealed that the genes encoding specific fungal secondary metabolites are clustered and often located near telomeres. In this review, we address some important questions, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

Relationship between Secondary Metabolism and Fungal Development

SUMMARY Filamentous fungi are unique organisms—rivaled only by actinomycetes and plants—in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway.

LaeA, a Regulator of Secondary Metabolism in Aspergillus spp.

ABSTRACT Secondary metabolites, or biochemical indicators of fungal development, are of intense interest to humankind due to their pharmaceutical and/or toxic properties. We present here a novel Aspergillus nuclear protein, LaeA, as a global regulator of secondary metabolism in this genus. Deletion of laeA (ΔlaeA) blocks the expression of metabolic gene clusters, including the sterigmatocystin (carcinogen), penicillin (antibiotic), and lovastatin (antihypercholesterolemic agent) gene clusters. Conversely, overexpression of laeA triggers increased penicillin and lovastatin gene transcription and subsequent product formation. laeA expression is negatively regulated by AflR, a sterigmatocystin Zn2Cys6 transcription factor, in a unique feedback loop, as well as by two signal transduction elements, protein kinase A and RasA. Although these last two proteins also negatively regulate sporulation, ΔlaeA strains show little difference in spore production compared to the wild type, indicating that the primary role of LaeA is to regulate metabolic gene clusters.

VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism.

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans, light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites.

Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis

SUMMARY Aspergillus species are globally ubiquitous saprophytes found in a variety of ecological niches. Almost 200 species of aspergilli have been identified, less than 20 of which are known to cause human disease. Among them, Aspergillus fumigatus is the most prevalent and is largely responsible for the increased incidence of invasive aspergillosis (IA) in the immunocompromised patient population. IA is a devastating illness, with mortality rates in some patient groups reaching as high as 90%. Studies identifying and assessing the roles of specific factors of A. fumigatus that contribute to the pathogenesis of IA have traditionally focused on single-gene deletion and mutant characterization. In combination with recent large-scale approaches analyzing global fungal responses to distinct environmental or host conditions, these studies have identified many factors that contribute to the overall pathogenic potential of A. fumigatus. Here, we provide an overview of the significant findings regarding A. fumigatus pathogenesis as it pertains to invasive disease.

Natural products of filamentous fungi: enzymes, genes, and their regulation

We review the literature on the enzymes, genes, and whole gene clusters underlying natural product biosyntheses and their regulation in filamentous fungi. We have included literature references from 1958, yet the majority of citations are between 1995 and the present. A total of 295 references are cited.

Aspergillus sporulation and mycotoxin production both require inactivation of the FadA Gα protein-dependent signaling pathway

The filamentous fungus Aspergillus nidulans contains a cluster of 25 genes that encode enzymes required to synthesize a toxic and carcinogenic secondary metabolite called sterigmatocystin (ST), a precursor of the better known fungal toxin aflatoxin (AF). One ST Cluster (stc) gene, aflR, functions as a pathway‐specific transcriptional regulator for activation of other genes in the ST pathway. However, the mechanisms controlling activation of aflR and synthesis of ST and AF are not understood. Here we show that one important level for control of stc gene expression requires genes that were first identified as early acting regulators of asexual sporulation. Specifically, we found that loss‐of‐function mutations in flbA, which encodes a RGS domain protein, or dominant activating mutations in fadA, which encodes the α subunit of a heterotrimeric G protein, block both ST production and asexual sporulation. Moreover, overexpression of flbA or dominant interfering fadA mutations cause precocious stc gene expression and ST accumulation, as well as unscheduled sporulation. The requirement for flbA in sporulation and ST production could be suppressed by loss‐of‐function fadA mutations. The ability of flbA to activate stc gene expression was dependent upon another early acting developmental regulator, fluG, and AflR, the stc gene‐specific transcription factor. These results are consistent with a model in which both asexual sporulation and ST production require inactivation of proliferative growth through inhibition of FadA‐dependent signaling. This regulatory mechanism is conserved in AF‐producing fungi and could therefore provide a means of controlling AF contamination.

Transcriptional Regulation of Chemical Diversity in Aspergillus fumigatus by LaeA

Secondary metabolites, including toxins and melanins, have been implicated as virulence attributes in invasive aspergillosis. Although not definitively proved, this supposition is supported by the decreased virulence of an Aspergillus fumigatus strain, ΔlaeA, that is crippled in the production of numerous secondary metabolites. However, loss of a single LaeA-regulated toxin, gliotoxin, did not recapitulate the hypovirulent ΔlaeA pathotype, thus implicating other toxins whose production is governed by LaeA. Toward this end, a whole-genome comparison of the transcriptional profile of wild-type, ΔlaeA, and complemented control strains showed that genes in 13 of 22 secondary metabolite gene clusters, including several A. fumigatus–specific mycotoxin clusters, were expressed at significantly lower levels in the ΔlaeA mutant. LaeA influences the expression of at least 9.5% of the genome (943 of 9,626 genes in A. fumigatus) but positively controls expression of 20% to 40% of major classes of secondary metabolite biosynthesis genes such as nonribosomal peptide synthetases (NRPSs), polyketide synthases, and P450 monooxygenases. Tight regulation of NRPS-encoding genes was highlighted by quantitative real-time reverse-transcription PCR analysis. In addition, expression of a putative siderophore biosynthesis NRPS (NRPS2/sidE) was greatly reduced in the ΔlaeA mutant in comparison to controls under inducing iron-deficient conditions. Comparative genomic analysis showed that A. fumigatus secondary metabolite gene clusters constitute evolutionarily diverse regions that may be important for niche adaptation and virulence attributes. Our findings suggest that LaeA is a novel target for comprehensive modification of chemical diversity and pathogenicity.

Histone deacetylase activity regulates chemical diversity in Aspergillus.

ABSTRACT Bioactive small molecules are critical in Aspergillus species during their development and interaction with other organisms. Genes dedicated to their production are encoded in clusters that can be located throughout the genome. We show that deletion of hdaA, encoding an Aspergillus nidulans histone deacetylase (HDAC), causes transcriptional activation of two telomere-proximal gene clusters—and subsequent increased levels of the corresponding molecules (toxin and antibiotic)—but not of a telomere-distal cluster. Introduction of two additional HDAC mutant alleles in a ΔhdaA background had minimal effects on expression of the two HdaA-regulated clusters. Treatment of other fungal genera with HDAC inhibitors resulted in overproduction of several metabolites, suggesting a conserved mechanism of HDAC repression of some secondary-metabolite gene clusters. Chromatin regulation of small-molecule gene clusters may enable filamentous fungi to successfully exploit environmental resources by modifying chemical diversity.