Anindya Chanda

A key role for vesicles in fungal secondary metabolism

Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B1; these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.

Compartmentalization and molecular traffic in secondary metabolism: A new understanding of established cellular processes

Great progress has been made in understanding the regulation of expression of genes involved in secondary metabolism. Less is known about the mechanisms that govern the spatial distribution of the enzymes, cofactors, and substrates that mediate catalysis of secondary metabolites within the cell. Filamentous fungi in the genus Aspergillus synthesize an array of secondary metabolites and provide useful systems to analyze the mechanisms that mediate the temporal and spatial regulation of secondary metabolism in eukaryotes. For example, aflatoxin biosynthesis in Aspergillus parasiticus has been studied intensively because this mycotoxin is highly toxic, mutagenic, and carcinogenic in humans and animals. Using aflatoxin synthesis to illustrate key concepts, this review focuses on the mechanisms by which sub-cellular compartmentalization and intra-cellular molecular traffic contribute to the initiation and completion of secondary metabolism within the cell. We discuss the recent discovery of aflatoxisomes, specialized trafficking vesicles that participate in the compartmentalization of aflatoxin synthesis and export of the toxin to the cell exterior; this work provides a new and clearer understanding of how cells integrate secondary metabolism into basic cellular metabolism via the intra-cellular trafficking machinery.

The initiation and pattern of spread of histone H4 acetylation parallel the order of transcriptional activation of genes in the aflatoxin cluster

The 27 genes involved in aflatoxin biosynthesis are clustered within a 70 kb region in the Aspergillus parasiticus genome. Using chromatin immunoprecipitation, we demonstrated a positive correlation between the initiation and spread of histone H4 acetylation in aflatoxin promoters and the onset of accumulation of aflatoxin proteins and aflatoxin. Histone H4 acetylation in the pksA (encodes an ‘early’ biosynthetic pathway enzyme) promoter peaked at 30 h, prior to the increased acetylation in the omtA and ordA (encode ‘late’ enzymes) promoters detected at 40 h. The specific order in which pksA, ver‐1 (encodes a ‘middle’ enzyme) and omtA transcripts accumulated in cells paralleled the pattern of spread of histone H4 acetylation. Binding of AflR, a positive regulator of aflatoxin biosynthesis, to the ordA promoter showed a positive correlation with the spread of histone H4 acetylation. The data suggest that the order of genes within the aflatoxin cluster determines the timing and order of transcriptional activation, and that the site of initiation and spread of histone H4 acetylation mediate this process. Our data indicate that the aflatoxin and adjacent sugar utilization clusters are part of a larger ‘regulatory unit’.

Stress-related Transcription Factor AtfB Integrates Secondary Metabolism with Oxidative Stress Response in Aspergilli

In filamentous fungi, several lines of experimental evidence indicate that secondary metabolism is triggered by oxidative stress; however, the functional and molecular mechanisms that mediate this association are unclear. The basic leucine zipper (bZIP) transcription factor AtfB, a member of the bZIP/CREB family, helps regulate conidial tolerance to oxidative stress. In this work, we investigated the role of AtfB in the connection between oxidative stress response and secondary metabolism in the filamentous fungus Aspergillus parasiticus. This well characterized model organism synthesizes the secondary metabolite and carcinogen aflatoxin. Chromatin immunoprecipitation with specific anti-AtfB demonstrated AtfB binding at promoters of seven genes in the aflatoxin gene cluster that carry CREs. Promoters lacking CREs did not show AtfB binding. The binding of AtfB to the promoters occurred under aflatoxin-inducing but not under aflatoxin-noninducing conditions and correlated with activation of transcription of the aflatoxin genes. Deletion of veA, a global regulator of secondary metabolism and development, nearly eliminated this binding. Electrophoretic mobility shift analysis demonstrated that AtfB binds to the nor-1 (an early aflatoxin gene) promoter at a composite regulatory element that consists of highly similar, adjacent CRE1 and AP-1-like binding sites. The five nucleotides immediately upstream from CRE1, AGCC(G/C), are highly conserved in five aflatoxin promoters that demonstrate AtfB binding. We propose that AtfB is a key player in the regulatory circuit that integrates secondary metabolism and cellular response to oxidative stress.

A Possible Role for Exocytosis in Aflatoxin Export in Aspergillus parasiticus

ABSTRACT Filamentous fungi synthesize bioactive secondary metabolites with major human health and economic impacts. Little is known about the mechanisms that mediate the export of these metabolites to the cell exterior. Aspergillus parasiticus synthesizes aflatoxin, a secondary metabolite that is one of the most potent naturally occurring carcinogens known. We previously demonstrated that aflatoxin is synthesized and compartmentalized in specialized vesicles called aflatoxisomes and that these subcellular organelles also play a role in the export process. In the current study, we tested the hypothesis that aflatoxisomes fuse with the cytoplasmic membrane to facilitate the release of aflatoxin into the growth environment. Microscopic analysis of A. parasiticus grown under aflatoxin-inducing and non-aflatoxin-inducing conditions generated several lines of experimental evidence that supported the hypothesis. On the basis of the evidence, we propose that export of the mycotoxin aflatoxin in Aspergillus parasiticus occurs by exocytosis, and we present a model to illustrate this export mechanism.

Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

BackgroundFilamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes.ResultsVolatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation.Conclusions1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary metabolites with catabolism of branched chain amino acids, alcohol biosynthesis, and β-oxidation of fatty acids. 3) Intracellular chemical development in A. parasiticus is linked to morphological development. 4) Understanding carbon flow through secondary metabolic pathways and catabolism of branched chain amino acids is essential for controlling and customizing production of natural products.

Proteomic and biochemical evidence support a role for transport vesicles and endosomes in stress response and secondary metabolism in Aspergillus parasiticus

Aflatoxin is among the most potent naturally occurring carcinogens known. Previous studies demonstrated that endosomes in the filamentous fungus Aspergillus parasiticus carry enzymes that catalyze the final two steps in aflatoxin synthesis, and these structures also play a role in aflatoxin storage and export. We hypothesized that endosomes house a complete and functional aflatoxin biosynthetic pathway. To address this hypothesis, we purified a cellular fraction containing endosomes, transport vesicles, and vacuoles (V fraction) from A. parasiticus grown under aflatoxin inducing and noninducing conditions. We also added (fed) aflatoxin pathway intermediates to V fraction to test the functional status of aflatoxin pathway enzymes. High throughput LC-MS/MS analysis of proteins in V fraction detected 8 aflatoxin enzymes with high reliability and 8 additional enzymes at lower reliability, suggesting that most aflatoxin pathway enzymes are present. Purified V fraction synthesized aflatoxin and addition of the pathway intermediate versicolorin A increased aflatoxin synthesis, confirming that middle and late aflatoxin enzymes in V fraction are functional. Of particular significance, proteomic and biochemical analysis strongly suggested that additional secondary metabolic pathways as well as proteins involved in response to heat, osmotic, and oxidative stress are housed in V fraction.

CYP2E1-dependent and leptin-mediated hepatic CD57 expression on CD8+ T cells aid progression of environment-linked nonalcoholic steatohepatitis.

Environmental toxins induce a novel CYP2E1/leptin signaling axis in liver. This in turn activates a poorly characterized innate immune response that contributes to nonalcoholic steatohepatitis (NASH) progression. To identify the relevant subsets of T-lymphocytes in CYP2E1-dependent, environment-linked NASH, we utilized a model of diet induced obese (DIO) mice that are chronically exposed to bromodichloromethane. Mice deficient in CYP2E1, leptin (ob/ob mice), or both T and B cells (Pfp/Rag2 double knockout (KO) mice) were used to delineate the role of each of these factors in metabolic oxidative stress-induced T cell activation. Results revealed that elevated levels of lipid peroxidation, tyrosyl radical formation, mitochondrial tyrosine nitration and hepatic leptin as a consequence of metabolic oxidative stress caused increased levels of hepatic CD57, a marker of peripheral blood lymphocytes including NKT cells. CD8+CD57+ cytotoxic T cells but not CD4+CD57+ cells were significantly decreased in mice lacking CYP2E1 and leptin. There was a significant increase in the levels of T cell cytokines IL-2, IL-1β, and IFN-γ in bromodichloromethane exposed DIO mice but not in mice that lacked CYP2E1, leptin or T and B cells. Apoptosis as evidenced by TUNEL assay and levels of cleaved caspase-3 was significantly lower in leptin and Pfp/Rag2 KO mice and highly correlated with protection from NASH. The results described above suggest that higher levels of oxidative stress-induced leptin mediated CD8+CD57+ T cells play an important role in the development of NASH. It also provides a novel insight of immune dysregulation and may be a key biomarker in NASH.

Quantitative Acoustic Contrast Tomography Reveals Unique Multiscale Physical Fluctuations during Aflatoxin Synthesis in Aspergillus parasiticus

Fungal pathogens need regulated mechanical and morphological fine-tuning for pushing through substrates to meet their metabolic and functional needs. Currently very little is understood on how coordinated colony level morphomechanical modifications regulate their behavior. This is due to an absence of a method that can simultaneously map, quantify, and correlate global fluctuations in physical properties of the expanding fungal colonies. Here, we show that three-dimensional ultrasonic reflections upon decoding can render acoustic contrast tomographs that contain information on material property and morphology in the same time scale of one important phytopathogen, Aspergillus parasiticus, at multiple length scales. By quantitative analysis of the changes in acoustic signatures collected as the A. parasiticus colony expands with time, we further demonstrate that the pathogen displays unique acoustic signatures during synthesis and release of its hepatocarcinogenic secondary metabolite, aflatoxin, suggesting an involvement of a multiscale morphomechanical reorganization of the colony in this process. Our studies illustrate for the first time, the feasibility of generating in any invading cell population, four-dimensional maps of global physical properties, with minimal physical perturbation of the specimens. Our developed method that we term quantitative acoustic contrast tomography (Q-ACT), provides a novel diagnostic framework for the identification of in-cell molecular factors and discovery of small molecules that may modulate pathogen invasion in a host.

Citrate-Coated Silver Nanoparticles Growth-Independently Inhibit Aflatoxin Synthesis in Aspergillus parasiticus

Manufactured silver nanoparticles (Ag NPs) have long been used as antimicrobials. However, little is known about how these NPs affect fungal cell functions. While multiple previous studies reveal that Ag NPs inhibit secondary metabolite syntheses in several mycotoxin producing filamentous fungi, these effects are associated with growth repression and hence need sublethal to lethal NP doses, which besides stopping fungal growth, can potentially accumulate in the environment. Here we demonstrate that citrate-coated Ag NPs of size 20 nm, when applied at a selected nonlethal dose, can result in a >2 fold inhibition of biosynthesis of the carcinogenic mycotoxin and secondary metabolite, aflatoxin B1 in the filamentous fungus and an important plant pathogen, Aspergillus parasiticus, without inhibiting fungal growth. We also show that the observed inhibition was not due to Ag ions, but was specifically associated with the mycelial uptake of Ag NPs. The NP exposure resulted in a significant decrease in transcript levels of five aflatoxin genes and at least two key global regulators of secondary metabolism, laeA and veA, with a concomitant reduction of total reactive oxygen species (ROS). Finally, the depletion of Ag NPs in the growth medium allowed the fungus to regain completely its ability of aflatoxin biosynthesis. Our results therefore demonstrate the feasibility of Ag NPs to inhibit fungal secondary metabolism at nonlethal concentrations, hence providing a novel starting point for discovery of custom designed engineered nanoparticles that can efficiently prevent mycotoxins with minimal risk to health and environment.