Mechanistic Insight on Vibrio Gazogenes Mediated Intervention of Aflatoxin Biosynthesis in Aspergillus Flavus

Aflatoxin is a liver carcinogen, mycotoxin, and a secondary metabolite that is produced by some plant pathogens within the genus Aspergillus when they infect crops such as corn, peanuts, and cotton. Given the significant adverse health and economic impacts of aflatoxin and a predicted rise of crop contamination with mycotoxins caused by a changing climate, there is a pressing need for exploring new aflatoxin mitigation methods. Previous studies in our laboratory have shown that metabolites from Vibrio gazogenes, an estuarine non-pathogenic bacterium, can inhibit aflatoxin synthesis. However, a direct interaction study between V. gazogenes and aflatoxin producing strains has not been conducted thus far. Here we show that administration of V. gazogenes in the growth medium of Aspergillus flavus, an aflatoxin producing pathogen in corn, results in an almost complete inhibition (\u3e99%) of aflatoxin biosynthesis at the level of gene expression. Light, electron and confocal microscopy suggested that V. gazogenes dependent aflatoxin inhibition was associated with internalization of bacterial cell materials but not intact bacteria, in endosome-like compartments. Administration of equal concentrations of heat-inactivated non-viable V. gazogenes cells to the growth medium resulted in a similar reduction in aflatoxin production, supporting that bacterial viability was not necessary for this inhibitory effect. We show that sclerotial development which depends on hyphal fusion decreased by more than 2-fold as well. Mycelia upon V. gazogenes administration showed early conidiation; however, conidia harvested from the treated colonies produced ~2-fold less aflatoxin than the earlier generation. We also show that, V. gazogenes uptake results in an almost complete (\u3e98%) block of hyphal fusion and an ~3-fold decrease of polar growth, processes that depend on endosomal functions, and transport. We have also found that A. flavus cannot uptake the same amount of V. gazogenes in the presence of endocytosis inhibitors which also affect the aflatoxin inhibition. In another study, we observed that, presence of A. flavus activates V. gazogenes lysing enzymes. Treating A. flavus with non-viable other gram-positive or gram-negative or prodigiosin producing bacterium did not have the same aflatoxin inhibitory effect. This indicates that the anti-aflatoxigenic activity is specific to V. gazogenes and prodigiosin is not the only aflatoxin inhibitor. Our study showed that, Intact V. gazogenes cells most effectively and efficiently decrease aflatoxin production (\u3e99%) rather than different fractionated parts of V. gazogenes cells. Collectively, our study introduces a novel cellular perturbation tool through V. gazogenes that can allow us to identify the interconnected molecular and cellular mechanisms that co-regulate secondary metabolism and hyphal development in filamentous fungi, hence providing a novel starting point for the discovery of a safe pharmaceutical and green fungicidal product that can efficiently prevent aflatoxins which have profound impacts on agriculture, environmental sustainability, and human health. The study ultimately unlocks an opportunity for furthering fundamental science and developing practically applicable strategies for intervening in Aspergillus toxin accumulation in the environment