Distribution and sub-cellular localization of the aflatoxin enzyme versicolorin B synthase in time-fractionated colonies of Aspergillus parasiticus

Abstract

Aflatoxins are highly toxic and carcinogenic fungal secondary metabolites. At least 18 enzyme activities are required for aflatoxin biosynthesis in the filamentous fungus Aspergillus parasiticus. One of these enzymes, versicolorin B synthase (VBS), catalyzes bisfuran ring closure in versiconal hemiacetal (a reaction near the middle of the pathway) to form versicolorin B. This reaction is required for the subsequent activation to aflatoxin B1-8,9 epoxide, a highly reactive and toxic aflatoxin metabolite, and is important for aflatoxin toxicity. We analyzed the localization of VBS in the aflatoxin-producing strain A. parasiticus SU-1 grown on solid media using a colony fractionation technique developed previously. A highly specific polyclonal antibody, raised against a maltose-binding protein-VBS fusion protein synthesized in Escherichia coli, was used to detect VBS in SU-1 grown on a rich solid medium via immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold transmission electron microscopy (TEM). VBS was detected in both vegetative hyphae and in asexual developmental structures, called conidiophores. Western blot and CLSM analyses demonstrated the highest abundance of VBS in colony fraction S2 consisting of cells that had grown for 24-48 h; this fraction also contained the highest levels of newly developed conidiophores and the highest abundance of aflatoxin B1, consistent with VBS abundance. At the subcellular level, CLSM and TEM detected VBS distributed throughout the cytoplasm and concentrated in ring-like structures surrounding nuclei. It is uncertain whether enzymatically active VBS is present in either or both locations.

Role of Oxidative Stress in Sclerotial Differentiation and Aflatoxin B1 Biosynthesis in Aspergillus flavus

“…Exported aflatoxin B1 levels were of the same magnitude with those measured when the fungus had been grown in a different version of the YES medium (25). Interestingly, A. flavus mycelial and exported aflatoxin B1 levels were similar to those in A. parasiticus (50). The transition of the control A. flavus from the undifferentiated (control-UD) to the sclerotial differentiated (control-SD) stage coincided with a 2-fold increase in the LOOH concentration.…”

A key role for vesicles in fungal secondary metabolism

“…The strains used in this study are listed in Table 1 : the model proposes that Nor-1 (early), Ver-1 (middle), and OmtA (late) are synthesized on free ribosomes in the cytoplasm, packaged into transport vesicles (6 -8), and transported to vacuoles via the cytoplasm-to-vacuole targeting (Cvt) pathway (6,8). In contrast Vbs localizes in the cytoplasm and in structures thought to be Golgi (29), suggesting it is transported to vacuoles via the classical secretory pathway; VBS is glycolsylated in support of this notion. Gene regulation (branch 2): sensing of glucose concentration or glucose metabolism initiates a FadA/cAMP/PKA signaling pathway (16); details of mechanism for sensing or metabolism of sucrose are not known.…”

Spatial and temporal control of fungal natural product synthesis

“…Versicolorin B synthase (Vbs), a late-pathway enzyme involved in aflatoxin biosynthesis also showed similar localization patterns. Vbs, the only glycosylated enzyme in the aflatoxin pathway, is observed to initially localize throughout the cytosol and then to ER-derived vesicles via the classical secretion mechanism 48 .…”