Regulation of Morphology, Aflatoxin Production, and Virulence of Aspergillus flavus by the Major Nitrogen Regulatory Gene areA.

Aspergillus flavus is a renowned plant, animal and human pathogen. areA is a global nitrogen regulatory gene of the GATA transcription factor family, shown to be the major nitrogen regulator. In this study, we identified areA in A. flavus and studied its function. The AreA protein contained a signatory zinc finger domain, which is extremely conserved across fungal species. Gene deletion (ΔareA) and over-expression (OE::areA) strains were constructed by homologous recombination to elucidate the role of areA in A. flavus. The ΔareA strain was unable to efficiently utilize secondary nitrogen sources for growth of A. flavus, and it had poorly developed conidiophores, when observed on complete medium, resulting in the production of significantly less conidia than the wild-type strain (WT). Aflatoxin B1 (AFB1) production was reduced in ΔareA compared with the WT strain in most conditions tested, and ΔareA had impaired virulence in peanut seeds. areA also played important roles in the sensitivity of A. flavus to osmotic, cell wall and oxidative stresses. Hence, areA was found to be important for the growth, aflatoxin production and pathogenicity of A. flavus. This work sheds light on the function of areA in the regulation of the nitrogen metabolism of A. flavus, and consequently aims at providing new ways for controlling the crossover pathogen, A. flavus.

Carbon catabolite repression gene creA regulates morphology, aflatoxin biosynthesis and virulence in Aspergillus flavus.

Carbon catabolite repression (CCR) is a very important mechanism employed in the utilization of carbon as an energy source, required for the regulation of growth, development and secondary metabolite production in fungi. Despite the wide study of this mechanism in fungi, little is known about the major CCR gene creA in A. flavus. Hence, we report identification of A. flavus carbon catabolite repression gene creA, which is responsible for the repression of secondary carbon sources. Gene deletion and over-expression was employed to explicate the role of creA in the morphology, pathogenicity, and secondary metabolite production in A. flavus. We investigated these factors using three carbon sources including glucose, sucrose and maltose. Gene deletion mutant (ΔcreA) had a significant growth defect on complete medium and minimal medium containing maltose. Conidia production in ΔcreA was significantly impaired irrespective of the carbon source available, while sclerotia production was significantly increased, compared to wild type (WT) and over-expression strain (OE::creA). Importantly, ΔcreA produced insignificant amount of aflatoxin in complete medium, and its ability to colonize hosts was also impaired. Concisely, we showed that creA played an important role in the morphology, pathogenicity and secondary metabolite production of A. flavus.

Effects of nitrogen metabolism on growth and aflatoxin biosynthesis in Aspergillus flavus.

Aflatoxins (AFs), produced mainly by Aspergillus flavus and Aspergillus parasiticus, are strongly toxic and carcinogenic. Here, we showed that glutamine is the optimal nitrogen source for AF-production in A. flavus grown in Czapek Dox medium. Additionally, 4mM glutamine was the threshold for high production of aflatoxin B1. However, no significant impact of glutamine synthetase inhibitor was detected for on AF biosynthesis. In contrast, rapamycin could significantly suppress the glutamine inducing effect on AFs production, simultaneously inhibiting the fungal growth and conidiation. To identify the genes and regulatory networks involved in AFs biosynthesis, especially concerning the nitrogen source metabolism pathway and the target of rapamycin (TOR) signaling pathway, we obtained transcriptomes for A. flavus under treatment of three nitrogen sources by RNA-sequencing. We identified 1429 differentially expressed genes. Through GO and KEGG pathway analyses, the relationship between nitrogen metabolism and AFs biosynthesis was revealed, and the effects of TOR inhibitor were confirmed. Additionally, the quantitative real-time PCR results verified the credibility and reliability of the RNA-seq data, and were consistent with the other experimental results. Our research laid the foundation for a primary study on the involvement of the nitrogen regulatory network and TOR signaling pathway in AF biosynthesis.

Functional Analysis of the Nitrogen Metabolite Repression Regulator Gene nmrA in Aspergillus flavus.

In Aspergillus nidulans, the nitrogen metabolite repression (NMR) regulator NmrA plays a major role in regulating the activity of the GATA transcription factor AreA during nitrogen metabolism. However, the function of nmrA in A. flavus has not been previously studied. Here, we report the identification and functional analysis of nmrA in A. flavus. Our work showed that the amino acid sequences of NmrA are highly conserved among Aspergillus species and that A. flavus NmrA protein contains a canonical Rossmann fold motif. Deletion of nmrA slowed the growth of A. flavus but significantly increased conidiation and sclerotia production. Moreover, seed infection experiments indicated that nmrA is required for the invasive virulence of A. flavus. In addition, the ΔnmrA mutant showed increased sensitivity to rapamycin and methyl methanesulfonate, suggesting that nmrA could be responsive to target of rapamycin signaling and DNA damage. Furthermore, quantitative real-time reverse transcription polymerase chain reaction analysis suggested that nmrA might interact with other nitrogen regulatory and catabolic genes. Our study provides a better understanding of NMR and the nitrogen metabolism network in fungi.

Aspergillus flavus SUMO Contributes to Fungal Virulence and Toxin Attributes.

Small ubiquitin-like modifiers (SUMOs) can be reversibly attached to target proteins in a process known as SUMOylation, and this process influences several important eukaryotic cell events. However, little is known regarding SUMO or SUMOylation in Aspergillus flavus. Here, we identified a novel member of the SUMO family in A. flavus, AfSumO, and validated the existence of SUMOylation in this pathogenic filamentous fungus. We investigated the roles of AfsumO in A. flavus by determining the effects of AfsumO mutations on the growth phenotype, stress response, conidia and sclerotia production, aflatoxin biosynthesis, and pathogenicity to seeds, and we found that SUMOylation plays a role in fungal virulence and toxin attributes. Taken together, these results not only reveal potential mechanisms of fungal virulence and toxin attributes in A. flavus but also provide a novel approach for promising new control strategies of this fungal pathogen.

Inhibition of aldose reductase ameliorates diet-induced nonalcoholic steatohepatitis in mice via modulating the phosphorylation of hepatic peroxisome proliferator-activated receptor α.

Aldose reductase (AR) is involved in the pathogenesis of nonalcoholic steatohepatitis. This study aimed to determine the mechanism by which AR affects the development of murine diet-induced nonalcoholic steatohepatitis. Steatohepatitis was induced in C57BL/6 mice by administration of a methionine-choline-deficient (MCD) diet, a commonly used nutrition-induced model of steatohepatitis. Hematoxylin and eosin staining was used for histological analyses. Western blot analyses were used to determine protein expression levels and quantitative polymerase chain reaction was used to analyze mRNA expression levels. The results showed that the AR protein expression level was significantly higher in C57BL/6 mice fed the MCD diet than in mice fed the control diet. Diet-induced hepatic steatosis and necroinflammation were attenuated in the MCD diet-fed mice treated with the AR inhibitor, zopolrestat. The ameliorating effect of AR inhibition on steatohepatitis was associated with decreased levels of serum alanine aminotransferase and hepatic lipoperoxides, reduced expression of phosphorylated peroxisome proliferator-activated receptor (PPAR)α and increased mRNA expression of acyl coenzyme A oxidase. These data indicated that induction of hepatic AR expression in mice with steatohepatitis resulted in the phosphorylation of PPARα and suppression of PPARα activity. Inhibition of AR decreased lipid accumulation and inflammation in the liver, at least in part through the modulation of PPARα phosphorylation and PPARα transcriptional activity.