Purification of a vesicle–vacuole fraction functionally linked to aflatoxin synthesis in Aspergillus parasiticus

Abstract

Current studies in our laboratory demonstrate a functional link between vesicles, vacuoles and aflatoxin biosynthesis in the filamentous fungus, Aspergillus parasiticus. Under aflatoxin inducing conditions in liquid yeast-extract sucrose medium, A. parasiticus undergoes a shift from vacuole biogenesis to accumulation of an enhanced number of vesicles which exhibit significant heterogeneity in size and density. As a first step in conducting a detailed analysis of the role of these organelles in aflatoxin synthesis, we developed a novel method to purify the vesicle and vacuole fraction using protoplasts prepared from cells harvested during aflatoxin synthesis. The method includes the following steps: 1] preparation of protoplasts from mycelia grown for 36h under aflatoxin inducing conditions; 2] release of vesicles and vacuoles from purified protoplasts in the presence of Triton X-100; and 3] fractionation of the vesicles and vacuoles using a “one-step high density cushion”. The vesicle-vacuole fraction showed a 35 fold enrichment in alpha-mannosidase activity (vacuole marker) and non-detectable succinate dehydrogenase and lactate dehydrogenase activities (mitochondrial and cytoplasmic markers, respectively). Confocal laser scanning microscopy with the vacuole dyes MDY-64 and CMAC demonstrated that the fraction contained pure vesicles and vacuoles and was devoid of membranous debris. Transmission electron microscopy (TEM) confirmed that no mitochondria or unbroken protoplasts contaminated the purified fraction. The purified organelles exhibited significant size heterogeneity with a range of sizes similar to that observed in whole cells and protoplasts.

A key role for vesicles in fungal secondary metabolism

“…The increased accumulation of Nor-1, Ver-1, Vbs, and OmtA levels that results from a block in the fusion of vesicles with vacuoles (current study) implies that all subsequent steps occur in af latoxisomes. Previous data from our lab and others strongly suggest that peroxisomes are not part of the purified vesiclevacuole fraction (12).…”

“…As a first step to determine the functional role of these organelles in Aspergillus secondary metabolism, we developed a unique ''high-density sucrose cushion'' method for purification of a vesicle-vacuole fraction from A. parasiticus during aflatoxin synthesis (12). In the current study, we show the functional role of this fraction in aflatoxin synthesis, storage, and export.…”

Identification of vacuole defects in fungi

“…Addition of polyvinylpyrrolidone can also be used to aid visualisation of Saccharomyces cerevisiae vacuoles (Gomes de Mesquita et al, 1991). Visualisation of vacuoles has also been studied by transmission electron microscopy (TEM; Chanda et al, 2009) and high resolution scanning electron microscopy (HRSEM; Isola et al, 2009). A B C Fig.…”

“…Cells are usually incubated with non-fluorescent compounds that, upon reaching the vacuole, undergo a chemical reaction generating a fluorescent derivative that is trapped in the vacuolar lumen. For example, chloromethyl coumarin-derived Cell Tracker vacuole markers, including CMAC (7-amino-4-chloromethylcoumarin; Molecular Probes) and CMFDA (5-chloromethylfluorescein diacetate), have been used to visualise vacuoles in a range of fungi such as Aspergillus nidulans (Leeder and Turner, 2008), Aspergillus parasiticus (Chanda et al, 2009), Aspergillus oryzae (Ohneda et al, 2005;Shoji et al, 2006;Tatsumi et al, 2007), U. maydis (Steinberg et al, 1998), Cryptococcus neoformans (Liu et al, 2006), Pisolithus tinctorius (Cole et al, 1997(Cole et al, , 1998 and Phanerochaete velutina (Zhuang et al, 2009). These compounds readily diffuse across cell membranes due to their hydrophobic nature.…”

“…This procedure has been used successfully to isolate vacuoles from yeast cells (Perzov et al, 2002;Ogita et al, 2010). A similar process can be used in filamentous fungi -for example protoplast formation followed by centrifugation on a high-density sucrose cushion has allowed vacuole isolation in A. parasiticus (Chanda et al, 2009). Purity of the isolated vacuole fraction was confirmed using specific markers for vacuoles, mitochondria and the cytosol and by microscopic methods (Chanda et al, 2009).…”