Proteomic and Biochemical Evidence Support a Role for Transport Vesicles and Endosomes in Stress Response and Secondary Metabolism in <i>Aspergillus parasiticus</i>

Aflatoxin is among the most potent naturally occurring carcinogens known. Previous studies demonstrated that endosomes in the filamentous fungus <i>Aspergillus parasiticus</i> 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 <i>A. parasiticus</i> 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

Proteomic and Biochemical Evidence Support a Role for Transport Vesicles and Endosomes in Stress Response and Secondary Metabolism in <i>Aspergillus parasiticus</i>

Aflatoxin is among the most potent naturally occurring carcinogens known. Previous studies demonstrated that endosomes in the filamentous fungus <i>Aspergillus parasiticus</i> 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 <i>A. parasiticus</i> 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

Aflatoxin levels in sunflower seeds and cakes collected from micro- and small-scale sunflower oil processors in Tanzania

<div><p>Aflatoxin, a mycotoxin found commonly in maize and peanuts worldwide, is associated with liver cancer, acute toxicosis, and growth impairment in humans and animals. In Tanzania, sunflower seeds are a source of snacks, cooking oil, and animal feed. These seeds are a potential source of aflatoxin contamination. However, reports on aflatoxin contamination in sunflower seeds and cakes are scarce. The objective of the current study was to determine total aflatoxin concentrations in sunflower seeds and cakes from small-scale oil processors across Tanzania. Samples of sunflower seeds (n = 90) and cakes (n = 92) were collected across two years, and analyzed for total aflatoxin concentrations using a direct competitive enzyme-linked immunosorbent assay (ELISA). For seed samples collected June-August 2014, the highest aflatoxin concentrations were from Dodoma (1.7–280.6 ng/g), Singida (1.4–261.8 ng/g), and Babati-Manyara (1.8–162.0 ng/g). The highest concentrations for cakes were from Mbeya (2.8–97.7 ng/g), Dodoma (1.9–88.2 ng/g), and Singida (2.0–34.3 ng/g). For seed samples collected August-October 2015, the highest concentrations were from Morogoro (2.8–662.7 ng/g), Singida (1.6–217.6 ng/g) and Mbeya (1.4–174.2 ng/g). The highest concentrations for cakes were from Morogoro (2.7–536.0 ng/g), Dodoma (1.4–598.4 ng/g) and Singida (3.2–52.8 ng/g). In summary, humans and animals are potentially at high risk of exposure to aflatoxins through sunflower seeds and cakes from micro-scale millers in Tanzania; and location influences risk.</p></div