Effects of laeA deletion on Aspergillus flavus conidial development and hydrophobicity may contribute to loss of aflatoxin production

LaeA of Aspergillus nidulans is a putative methyltransferase and a component of the velvet complex; it is thought to mainly affect expression of genes required for the production of secondary metabolites. We found that although Aspergillus flavus CA14 laeA deletion mutants showed no aflatoxin production, expression of some of the early genes involved in aflatoxin formation, but not the later genes, could still be detected. The mutants grown in minimal medium supplemented with simple sugars and on some complex media exhibited altered conidial development. On potato dextrose agar (PDA) medium the deletion mutants showed reduced conidial chain elongation, increased production of conidiophores, and decreased colony hydrophobicity when compared to the parental strain. The loss of hydrophobicity and the other developmental changes in the laeA deletion mutants could affect the ability of the fungus to produce aflatoxins.     

Aspergillus oryzae laeA regulates kojic acid synthesis genes

Kojic acid synthesis genes regulation was investigated in Aspergillus oryzae. Our results indicate that kojic acid production was lost in the laeA disruption strain, but was recovered in the LaeA complement strain. Real-time PCR also confirmed that expression of kojic acid biosynthesis genes decreased in the laeA disruption strain, indicating that these genes are under the control of LaeA.     

LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity

VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells), which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators.     

Beyond aflatoxin: four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters

Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis has predicted that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in Aspergillus flavus ; however, only three metabolic pathways—aflatoxin, cyclopiazonic acid (CPA) and aflatrem—have been assigned to these clusters. To gain an insight into the regulation of and to infer the ecological significance of the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture medium and temperature, fungal development, colonization of developing maize seeds and misexpression of laeA , a global regulator of secondary metabolism. Hierarchical clustering analysis of expression profiles allowed us to categorize the gene clusters into four distinct clades. Gene clusters for the production of aflatoxins, CPA and seven other unknown compound(s) were identified as belonging to one clade. To further explore the relationships found by gene expression analysis, aflatoxin and CPA production were quantified under five different cell culture environments known to be conducive or nonconducive for aflatoxin biosynthesis and during the colonization of developing maize seeds. Results from these studies showed that secondary metabolism gene clusters have distinctive gene expression profiles. Aflatoxin and CPA were found to have unique regulation, but are sufficiently similar that they would be expected to co-occur in substrates colonized with A. flavus .     

丝状真菌新型全局性调控因子LaeA

全局调控在丝状真菌次级代谢调控及其生长发育过程中有着重要的作用。LaeA是2004年首次在构巢曲霉中被发现的第一个丝状真菌全局性调控因子,继而在烟曲霉、黄曲霉、产黄青霉、橘青霉中相继被报道。LaeA能够全局性调控抗生素和真菌毒素等次级代谢产物的合成,影响真菌形态分化,另外还通过影响沉默基因的表达从而调控未知代谢产物的产生,因而能为真菌中天然产物的开发提供新的重要途径。本文就其在丝状真菌中的发现、功能、作用机制及其应用等方面进行综述。     

The global regulator LaeA controls production of citric acid and endoglucanases in <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis>

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74–96 % compared to the wild type. The endoglucanase activity was reduced with 51–78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.     

Fungal Secondary Metabolites and Their Fundamental Roles in Human Mycoses

Understanding which fungal factors allow colonization and infection of a human host is critical to lowering the incidence of human mycoses and related mortalities. In the pathogen Aspergillus fumigatus, secondary metabolites, small bioactive molecules produced by many opportunistic fungal pathogens, have important roles in suppressing and providing protection from host defenses. Deletion of LaeA, a global regulator of secondary metabolism in fungi, significantly decreases A. fumigatus virulence, in part owing to loss of gliotoxin and hydrophobin production. In addition to gliotoxin, dihydroxynaphthalene (DHN) melanin and siderophores are other A. fumigatus virulence factors; all three metabolites are derived from hallmark secondary metabolite gene clusters. Many of the gene clusters producing toxin metabolites have yet to be deciphered, and the study of secondary metabolites and their role in the virulence of human pathogens is a nascent field.     

The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum

The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.     

LaeA regulation of secondary metabolism modulates virulence in Penicillium expansum and is mediated by sucrose

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose‐responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175‐ and five‐fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe‐21 from Israel and Pe‐T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch‐Pe‐T01 strains to 15%–25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is‐Pe‐21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.     

Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>: cross-talk regulation of secondary metabolite pathways

Penicillium chrysogenum is an excellent model fungus to study the molecular mechanisms of control of expression of secondary metabolite genes. A key global regulator of the biosynthesis of secondary metabolites is the LaeA protein that interacts with other components of the velvet complex (VelA, VelB, VelC, VosA). These components interact with LaeA and regulate expression of penicillin and PR-toxin biosynthetic genes in P. chrysogenum. Both LaeA and VelA are positive regulators of the penicillin and PR-toxin biosynthesis, whereas VelB acts as antagonist of the effect of LaeA and VelA. Silencing or deletion of the laeA gene has a strong negative effect on penicillin biosynthesis and overexpression of laeA increases penicillin production. Expression of the laeA gene is enhanced by the P. chrysogenum autoinducers 1,3 diaminopropane and spermidine. The PR-toxin gene cluster is very poorly expressed in P. chrysogenum under penicillin-production conditions (i.e. it is a near-silent gene cluster). Interestingly, the downregulation of expression of the PR-toxin gene cluster in the high producing strain P. chrysogenum DS17690 was associated with mutations in both the laeA and velA genes. Analysis of the laeA and velA encoding genes in this high penicillin producing strain revealed that both laeA and velA acquired important mutations during the strain improvement programs thus altering the ratio of different secondary metabolites (e.g. pigments, PR-toxin) synthesized in the high penicillin producing mutants when compared to the parental wild type strain. Cross-talk of different secondary metabolite pathways has also been found in various Penicillium spp.: P. chrysogenum mutants lacking the penicillin gene cluster produce increasing amounts of PR-toxin, and mutants of P. roqueforti silenced in the PR-toxin genes produce large amounts of mycophenolic acid. The LaeA-velvet complex mediated regulation and the pathway cross-talk phenomenon has great relevance for improving the production of novel secondary metabolites, particularly of those secondary metabolites which are produced in trace amounts encoded by silent or near-silent gene clusters.     

Effect of the raw extracts of Arthrinium strains (Hyphomycetes, Dematiaceae) on the growth of some deleterious fungi in poultry feed

In previous work the authors have shown that some species of the Arthrinium genus are characterized by being able to produce secondary metabolites with antibiotic activity. The aim of this study was to evaluate the effect of raw extracts of the growth of three different Arthrinium strains against Aspergillus flavus, Aspergillus nidulans, Fusarium moniliforme and Penicillium purpurogenum when they were present in poultry feed. The results showed that the extracts reduced the growth of Aspergillus flavus and Fusarium moniliforme but could not inhibit the development of Aspergillus nidulans. Only the raw extract of A. aureum inhibited the growth of Penicillium purpurogenum.