Jay E. Mellon

Aspergillus flavus hydrolases : their roles in pathogenesis and substrate utilization

Aspergillus flavus is a fungus that principally obtains resources for growth in a saprophytic mode. Yet, it also possesses the characteristics of an opportunistic pathogen with a wide, non-specific host range (plants, animals, and insects). It has attained a high level of agricultural significance due to production of the carcinogen aflatoxin, which significantly reduces the value of contaminated crops. To access a large variety of nutrient substrates and penetrate host tissues, A. flavus possesses the capacity to produce numerous extracellular hydrolases. Most work on A. flavus hydrolases has focused on the serine and metalloproteinases, pectinase P2c, and amylase. Many hydrolases are presumed to function in polymer degradation and nutrient capture, but the regulation of hydrolase secretion is complex and substrate dependent. Proteinases are employed not only to help access protein substrates, such as elastin that is found in mammals and insects, but may also play roles in fungal defense and virulence. Secretion of the endopolygalacturonase P2c is strongly correlated with isolate virulence (against plants) and maceration of cotton boll tissues. In some hosts, secretion of α-amylase is critical for starch digestion and may play a critical role in induction of aflatoxin biosynthesis. Despite a significant body of work, much remains to be learned about hydrolase production and utilization by A. flavus. This information may be critical for the formulation of successful strategies to control aflatoxin contamination in affected commodities.

Expression of pectinase activity among Aspergillus flavus isolates from southwestern and southeastern United States

Aspergillus flavus is a widely distributed filamentousfungus that contaminates crops with the potent carcinogenaflatoxin. This species can be divided into S and L strainson the basis of sclerotial morphology. During crop infection,A. flavus can secrete a large array of hydrolytic enzymes.These include pectinase, which aids fungal spread throughplant tissues. A survey of pectinase expression by soilisolates derived from different regions of the UnitedStates revealed geographic polymorphisms. Strain Lisolates from Arizona produced moderate to high levelsof a specific pectinase P2c, while S strain isolates producedvariable amounts of P2c. In contrast, L strain isolates fromsoutheastern U.S. yielded variable P2c production, while Sstrain isolates consistently expressed high P2c levels.These results were corroborated by pectinase surveys ofadditional collections of A. flavus from soil and cottonseed.Expression patterns for P2c and pectinmethylesterase wereevaluated for a select number of isolates using an isoelectricfocusing technique. Clear zone reactions from the pectinaseplate assay corresponded to the presence of P2c, while redring reactions corresponded to the lack of P2c. Commercialcottonseed infected by S strain isolates frequently containedaflatoxin, even when infected by S strain isolates that didnot produce pectinase P2c. Thus, although P2c-lackingisolates have reduced invasiveness, these isolates still havesufficient pathogenicity to cause aflatoxin contamination.

Inhibitory effects of gossypol, gossypolone, and apogossypolone on a collection of economically important filamentous fungi.

Racemic gossypol and its related derivatives gossypolone and apogossypolone demonstrated significant growth inhibition against a diverse collection of filamentous fungi that included Aspergillus flavus, Aspergillus parasiticus, Aspergillus alliaceus, Aspergillus fumigatus, Fusarium graminearum, Fusarium moniliforme, Penicillium chrysogenum, Penicillium corylophilum, and Stachybotrys atra. The compounds were tested in a Czapek agar medium at a concentration of 100 μg/mL. Racemic gossypol and apogossypolone inhibited growth by up to 95%, whereas gossypolone effected 100% growth inhibition in all fungal isolates tested except A. flavus. Growth inhibition was variable during the observed time period for all tested fungi capable of growth in these treatment conditions. Gossypolone demonstrated significant aflatoxin biosynthesis inhibition in A. flavus AF13 (B(1), 76% inhibition). Apogossypolone was the most potent aflatoxin inhibitor, showing greater than 90% inhibition against A. flavus and greater than 65% inhibition against A. parasiticus (B(1), 67%; G(1), 68%). Gossypol was an ineffectual inhibitor of aflatoxin biosynthesis in both A. flavus and A. parasiticus. Both gossypol and apogossypolone demonstrated significant inhibition of ochratoxin A production (47%; 91%, respectively) in cultures of A. alliaceus.

Purification and characterization of anionic peroxidases from cotton (Gossypium hirsutum L.)

Abstract Anionic isoperoxidases from cotton cotyledonary leaf tissue have been purified to electrophoretic homogeneity by chromatography on ion exchange, lectin affinity, and gel filtration matrices. The enzyme was stable and active at a wide range of temperatures with optimal activity at 55°C. Maximum activity was observed when the pH was adjusted from pH 5.5 to pH 7.5. Electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels indicated a relative molecular mass of 56 000 Da when the sample was treated with a disulfide reducing agent and heated. The electrophoretic mobility was altered when either the disulfide reducing agent or heat treatment was omitted. Isoelectric focusing under native conditions resolved the activity into three isozymes with isoelectric points of 4.2, 4.4 and 4.6. The K m of peroxidase for 4-aminoantipyrine and o -dianisidine was 350 and 36 μM, respectively. Enzyme activity was inhibited by elevated concentrations of H 2 O 2 and by disulfide reducing agents. Known inhibitors of peroxidase activity such as potassium azide, sodium cyanide and sodium sulfite inhibited cotton anionic peroxidases. The absorption spectrum showed maxima at 280 and 402 nm, indicating the presence of a heme group in the active enzyme. The extinction coefficient at 402 nm was 1.12 × 10 5 mol −1 cm −1 .

Purification and partial characterization of an elastinolytic proteinase from Aspergillus flavus culture filtrates

Abstract A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene (5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0. Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein, collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range of potential natural protein substrates.

Effects of oilseed storage proteins on aflatoxin production by Aspergillus flavus

Potential involvement of seed storage proteins in susceptibility to aflatoxin contamination was assessed with in vitro tests. Initially, two oilseed storage proteins [cottonseed storage protein (CSP) and zein] were compared with bovine serum albumin (BSA) and collagen. Supplementation of a complete defined medium with either oilseed storage protein resulted in significantly more aflatoxin production by Aspergillus flavus than supplementation with either BSA or collagen. Little or no aflatoxin was produced when either BSA, CSP, or zein was employed (at 0.5%) as both the sole carbon and the sole nitrogen source. Media with collagen (0.5%) as the sole nitrogen and carbon source supported aflatoxin production similar to the complete defined medium. Although lower than levels observed with defined medium, aflatoxin production increased with both increasing CSP concentration (0 to 2.0%) and increasing zein concentration (0 to 6.0%) when these proteins served as both the sole carbon and sole nitrogen source. Denaturing polyacrylamide gel electrophoresis and protease activity assays indicated that fungal acquisition of protein carbon was probably via hydrolysis mediated by the 35 kD metalloprotease of A. flavus. Media lacking nitrogen but containing sucrose (5.0%) and supplemented with either zein (1.7%) or CSP (2.0%) supported three- to eightfold more aflatoxin production than the complete defined medium. The results suggest seed storage proteins, when present with an accessible carbon source, may predispose oilseed crops to support production of high levels of aflatoxins by A. flavus during seed infection.

Inhibition of aflatoxin production inAspergillus flavus by cotton ovule extracts

The effect of cotton ovule extracts onAspergillus flavus growth and aflatoxin biosynthesis was investigated. Extracts derived from either noninoculated cotton ovule tissue or cotton ovule tissue challenged withA. flavus inhibited aflatoxin (B1) synthesis by fungal cultures ofA. flavus. When added to fungal cultures in concentrations of 50 µg per mL of medium, extracts derived from fungus-challenged ovule tissue inhibited aflatoxin synthesis by as much as 93%. The ED50 for this type of extract was 18 µg per mL of medium (P < 0.01; r2=0.46). Similar experiments with a noninoculated ovule extract (50 µg/mL medium) produced aflatoxin inhibition levels of up to 77%. The ED50 for noninoculated extracts was 35 µg per mL of medium (P < 0.01; r2=0.66). These extracts did not inhibit the growth ofA. flavus in culture. Cultures ofA. flavus that contained pure gossypol (10–50 µg/mL medium) also showed significantly reduced levels of aflatoxin production. Gossypol may account for the aflatoxin-inhibitory activity observed in the extracts derived from noninoculated cotton ovule tissue.