Project description:Mycotic leratitis is the corneal inflammation predominantly caused by Fusarium and Aspergillus species. Corneal epithelium is the earliest cell type encounter the invading pathogen. The innate immune responses of human corneal epithelial cells against Aspergillus flavus is not known. Here we studied the role of human corneal epithelial cells against Aspergillus flavus infection. The results showed that corneal epithelial cells internalized Aspergillus flavus conidia through actin mediated polymerization surrounding the conidia. Further the actin inhibitor cytochalasin D treatment reduced the formation actin ring around the conidia. The engulfed conidia acquired endosomal proteins as revealed by immunofluorescence analsyisis. Mass spectromtery of phagosomal proteins confirmed the recruitment of endosomal proteins and other proteins involved in phagocytosis. These results show the involvement of corneal epithelial cells in anti fungal defense.
Project description:Aspergillus flavus and A. parasiticus are two of the most important aflatoxin-producing species that contaminate agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here, we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O-methylsterigmatocystin, but not CPA. Only four out of forty-five attempted interspecific crosses between compatible mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were isolated and were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of putative allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important species.
Project description:Objective: Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of GO analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. This study aimed to investigate the integrative function of the aflR gene in A. flavus. Design: In this study, we used Aspergillus flavus NRRL3357 as a wild-type strain (WT) and constructed a knockout strain of A. flavus ΔaflR by homologous recombination. Based on the transcriptomics technology, we investigated the metabolic effects of aflR gene on growth, development and toxin synthesis of A. flavus, and discussed the overall regulation mechanism of aflR gene on A. flavus at the transcriptional level. Results: The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. In addition, disrupted strains of the aflR gene produced relatively sparse conidia and a very small number of sclerotia. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly down-regulated. Meanwhile, the ΔaflR strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT strain. Conclusions: The results showed that the A. flavus aflR gene also played a positive role in the growth and development of fungi.
Project description:Linking cell reproduction and survival is a key task of all life forms. All fungi in the genus Aspergillus reproduce by forming asexual spores called conidia, of which formation is governed by the central regulatory circuit, BrlA->AbaA->WetA. Here, we report that WetA is a key multi-functional regulator that bridged spore differentiation, long-term survival, and chemical development in Aspergillus flavus.
Project description:Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. In order to better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. A. flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B1 and B2 biosynthesis, while A. parasiticus cultures had significantly increased B1 and G1 biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed seventy seven genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis. Keywords: Aflatoxin, Aspergillus, flavus, Amnio Acids, Tryptophan
Project description:Human corneal epithelial cells internalize Aspergillus flavus conidia. But the biochemical response of corneal epithelial cells in response to A.flavus is not clear. In this study, NanoString analysis was used to study the immune genes expression in primary corneal epithelial cells and cell line infected with A. flavus conidia. The study revealed the differential expression of immune genes associated with TNF, Th17, NF-kB signaling and B cell receptor signaling in corneal epithelial cells. There is also difference in the response between cell line and primary cells to A. flavus implies the use of both cells to understand the host-pathogen interaction.
Project description:RNA-seq was used to compare differential gene expressions for Aspergillus flavus wild type strain and ASPES transcription factor deletion strains.The goals of this study are to explore the aflatoxin regulation pathway in A. flavus.