Date of Award

2018

Document Type

Open Access Dissertation

Department

Environmental Health Sciences

First Advisor

Anindya Chanda

Abstract

Aflatoxin, a mycotoxin, is one of the world’s most potent carcinogen. It contaminates major food products such as milk, grains, nuts, corn, etc., leading to greater than $ 1 billion in economic losses and when ingested causes hepatocellular carcinoma (HCC). It is the primary risk factor for 75% HCC cases in the developing world and 3% HCC cases in developed world. Most common methods used in agriculture to reduce aflatoxin contamination are expensive, time consuming and have low efficiency with limited success rates where as biological controls were proven to be most effective in inhibiting aflatoxins and aflatoxin producing fungi. Vibrio gazogenes, a non-pathogenic gram-negative marine bacterium, was proven to synthesize antifungal and antiaflatoxin metabolites. In this research study we have used Aspergillus parasiticus and Aspergillus flavus – two saprophytic pathogenic fungi as aflatoxin-producing models. Preliminary experimentation by treating V. gazogenes with aflatoxin produced aflatoxin responsive metabolites (ARMs) that had the ability to significantly decrease aflatoxin synthesis by inhibiting the aflatoxin genes (aflR, nor-1, ver-1) and global secondary metabolism genes (LaeA, VeA). But the decrease in aflatoxin was only 40%. So we treated the fungal cultures with the cells of V. gazogenes and the aflatoxin ELISAs revealed the significant decrease (>99%) in aflatoxin biosynthesis by the fungi. The aflatoxin inhibitory effect was very specific to V. gazogenes and not to other gram-positive or gram-negative bacterium. Infecting corn kernels with A. flavus in the presence of bacterium significantly decrease the fungal conidial growth by 50% and aflatoxin by 98%. Treating drosophila vi flies with V. gazogenes prior to A. flavus infection increased their survival. Using confocal, scanning electron and transmission electron microscopies we observed the uptake of the bacterium by the fungus into vesicles. RT-PCR assays revealed that live V. gazogenes cells significantly up-regulate aflatoxin genes (aflR, nor-1, ver-1) and global secondary metabolite genes (laeA, veA). The pathway through which V. gazogenes inhibits aflatoxin is complicating. But our study had clearly developed a novel tool (V. gazogenes) to inhibit the aflatoxin biosynthesis, which is acting at the cellular level rather than at the gene level.

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