Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces

Abstract

The aim of this study is to investigate aflatoxin gene expression during Streptomyces-Aspergillus interaction. Aflatoxins are carcinogenic compounds produced mainly by Aspergillus flavus and Aspergillus parasiticus. A previous study has shown that Streptomyces-A. flavus interaction can reduce aflatoxin content in vitro. Here, we first validated this same effect in the interaction with A. parasiticus. Moreover, we showed that growth reduction and aflatoxin content were correlated in A. parasiticus but not in A. flavus. Secondly, we investigated the mechanisms of action by reversetranscriptase quantitative PCR. As microbial interaction can lead to variations in expression of household genes, the most stable [act1, btub (and cox5 for A. parasiticus)] were chosen using geNorm software. To shed light on the mechanisms involved, we studied during the interaction the expression of five genes (aflD, aflM, aflP, aflR and aflS). Overall, the results of aflatoxin gene expression showed that Streptomyces repressed gene expression to a greater level in A. parasiticus than in A. flavus. Expression of aflR and aflS was generally repressed in both Aspergillus species. Expression of aflM was repressed and was correlated with aflatoxin B1 content. The results suggest that aflM expression could be a potential aflatoxin indicator in Streptomyces species interactions. Therefore, we demonstrate that Streptomyces can reduce aflatoxin production by both Aspergillus species and that this effect can be correlated with the repression of aflM expression. INTRODUCTIONAflatoxins (AFs) are polyketide-derived furanocoumarins. They are produced by fungi of the genus Aspergillus (including Aspergillus flavus and Aspergillus parasiticus) in agricultural foodstuffs (maize, hazelnut, peanut, etc.) (Giorni et al., 2007;Passone et al., 2010). These AFs are toxic and their main adverse effects on humans are hepatocarcinoma (Qian et al., 1994; IARC, 2014), immune system deficiency (Jiang et al., 2005), reduced child growth (Gong et al., 2004) and increased risks of stillborn or newborn jaundice (Shuaib et al., 2010). To reduce those multiple effects, many countries have implemented maximum authorized levels of AFs in food and feed (Wu & Guclu, 2012). AF biosynthesis is coded by a 80 kb long DNA sequence. The latter is a cluster containing 30 putative genes characterized in both A. flavus and A. parasiticus (Yu, 2012). For structural genes, early (as aflD), medium (as aflM) and late (as aflP) genes are denominated (Fig. S1, available in the online Supplementary Material). The gene aflD encodes a reductase enzyme involved in the conversion of norsolorinic acid to averantin (Papa, 1982); aflM is required for the conversion of versicolorin A to demethylsterigmatocystin (Skory et al., 1992); and aflP encodes a methyltransferase converting sterigmatocystin to O-methylsterigmatocystin (Bhatnagar et al., 1988). Two cluster-specific regulators are also known: aflR encodes a transcription activator that binds a consensus sequence in the promoter regions of AF ...

Dual Transcriptional Profile of Aspergillus flavus during Co-Culture with Listeria monocytogenes and Aflatoxin B1 Production: A Pathogen–Pathogen Interaction

“…In fact, in our experiments, we did not observe any statistically significant change in A. flavus biomass during co-culture with L. monocytogenes (data not shown). This observation is in accordance with Verheecke et al [48], reporting that growth was not correlated with AFB1 in A. flavus during co-culture conditions. Furthermore, in the present study, the influence of this co-existence in A. flavus relative gene expression was also assessed.…”

“…At the same time, the downregulation of aflM and aflP during monoculture, as well as aflM and aflS during co-culture, were observed. These results are also in concordance with Verheecke et al [48] who similarly observed that the expression of aflM was repressed by Streptomyces presence and was also correlated with AFB1 content. The complex influence that temperature may have on the regulation of A. flavus aflatoxin biosynthetic pathway has been previously demonstrated [14,53].…”

“…However, in the present work, a significant reduction of AFB1 was evident after co-culture with L. monocytogenes. In general, the regulatory system of fungal secondary metabolites production can be influenced by a wide range of parameters which are not always associated with growth [48,49]. In fact, in our experiments, we did not observe any statistically significant change in A. flavus biomass during co-culture with L. monocytogenes (data not shown).…”

Peer Review #2 of "Oxidative stress and the presence of bacteria increase gene expression of the antimicrobial peptide aclasin, a fungal CSαβ defensin in Aspergillus clavatus (v0.2)"

“…Quantitative real-time PCR (qPCR) was carried out in triplicate in a LightCycler 96 instrument (Roche, Germany) using 100 ng cDNA, 0.5 µM each primer (Table S1) and qPCR BIO SyGreen Mix Lo-ROX (PCR Biosystem, UK) in 10 µl of total volume. The act1 gene (ACLA_095800) was used as reference gene (Bohle et al, 2007;Verheecke et al, 2015). Threshold cycle (C t ) determination was performed using LightCycler 96 software (Roche, Germany).…”

Microbe-Mediated Control of Mycotoxigenic Grain Fungi in Stored Rice with Focus on Aflatoxin Biodegradation and Biosynthesis Inhibition

“…However, to better understand how microbe-related inhibition of aflatoxin occurs, the aflatoxin-biosynthetic processes and the related genes will be discussed. To this end, there are interesting studies on the possible mechanisms responsible for the down-regulation of the transcription-activator genes aflS (formerly aflJ ) and aflR [ 75 103 ] in Aspergillus spp. Changes in the aflR / aflS -expression ratio in response to temperature and water activity have also been reported [ 102 ].…”