Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus

Abstract

The activities of two enzymes, a 168-kDa protein and a 40-kDa protein, OmtA, purified from the filamentous fungus Aspergillus parasiticus were reported to convert the aflatoxin pathway intermediate sterigmatocystin to O-methylsterigmatocystin in vitro. Our initial goal was to determine if OmtA is necessary and sufficient to catalyze this reaction in vivo and if this reaction is necessary for aflatoxin synthesis. We generated A. parasiticus omtA-null mutant LW1432 and a maltose binding protein-OmtA fusion protein expressed in Escherichia coli. Enzyme activity analysis of OmtA fusion protein in vitro confirmed the reported catalytic function of OmtA. Feeding studies conducted with LW1432 demonstrated a critical role for OmtA, and the reaction catalyzed by this enzyme in aflatoxin synthesis in vivo. Because of a close regulatory link between aflatoxin synthesis and asexual sporulation (conidiation), we hypothesized a spatial and temporal association between OmtA expression and conidiospore development. We developed a novel time-dependent colony fractionation protocol to analyze the accumulation and distribution of OmtA in fungal colonies grown on a solid medium that supports both toxin synthesis and conidiation. OmtA-specific polyclonal antibodies were purified by affinity chromatography using an LW1432 protein extract. OmtA was not detected in 24-h-old colonies but was detected in 48-h-old colonies using Western blot analysis; the protein accumulated in all fractions of a 72-h-old colony, including cells (0 to 24 h) in which little conidiophore development was observed. OmtA in older fractions of the colony (24 to 72 h) was partly degraded. Fluorescence-based immunohistochemical analysis conducted on thin sections of paraffin-embedded fungal cells from time-fractionated fungal colonies demonstrated that OmtA is evenly distributed among different cell types and is not concentrated in conidiophores. These data suggest that OmtA is present in newly formed fungal tissue and then is proteolytically cleaved as cells in that section of the colony age.

Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein

“…Ver-1 (unlike another focus of our studies, the middle AF pathway enzyme VBS) does not carry typical glycosylation motifs, strongly suggesting it is not transported by the secretory pathway. In addition, we have noted in several studies that Ver-1 protein (as well as Nor-1 and OmtA) is subject to proteolytic cleavage at a single site and the level of cleavage appears to increase in parallel with the rate of AF synthesis (22,25,44). Based on these observations, we hypothesize that Ver-1 utilizes the Cvt pathway for vacuolar localization (see the model in Fig.…”

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

“…E. coli DH5␣ carrying pMBP-AtfB was incubated in 5 ml of Luria-Bertani broth containing ampicillin (100 g per ml) for 16 h. One ml of bacterial culture was saved as uninduced control. The remaining 4 ml of culture was induced to express fusion protein by the addition of 0.3 mM isopropyl ␤-D-thiogalactopyranoside for 3 h. Large scale production and purification of MBP::AtfB fusion protein were conducted (amylose affinity column chromatography) using methods described previously (46).…”

A Novel cAMP-response Element, CRE1, Modulates Expression of nor-1 in Aspergillus parasiticus

“…Nor-1-maltose-binding protein (7) was used as antigen for polyclonal antibody production in rabbits using procedures published previously for anti-Ver-1 (10) and anti-OmtA (45). In YES liquid shake cultures, the antibody (IgG fraction) detected two major bands at 31 and 28 kDa at 48 and 60 h in A. parasiticus SU-1; neither band was detected in a Nor-1-disrupted strain (⌬nor-1) (8) at any time point (not shown).…”