The loss of the inducible Aspergillus carbonarius MFS transporter MfsA leads to ochratoxin A overproduction

Ochratoxin A (OTA), a nephrotoxic compound produced by certain Aspergillus and Penicillium species, is one of the most abundant mycotoxins in food commodities. Aspergillus carbonarius is the main source of OTA in wine, grape juice and dried vine fruits. Although many studies have focused on OTA production by A. carbonarius, little is known about the genes related to OTA production and transport. We have found a transporter that belongs to the major facilitator superfamily (MfsA) which is highly expressed with a 102-fold induction in an ochratoxigenic A. carbonarius strain compared to a low OTA producer strain. The encoding mfsA gene shows similarity to the multidrug efflux transporter flu1 from Candida albicans. A high number of putative transcription factor binding sites involved in the response to stress were identified within the promoter of mfsA. Phenotypical analysis of δ. mfsA deletion mutants revealed that the loss of mfsA leads to a slight growth reduction and increased OTA production. We therefore hypothesize that MfsA could be a stress response transporter whose disruption could cause an increase in oxidative stress together with a stimulation of mycotoxin production. •A. carbonarius mfsA expression is induced over 100 fold in an OTA producing strain.•mfsA gene has been cloned and sequenced.•An A. carbonarius δmfsA null mutant has been obtained by ATMT.•The δmfsA null mutants produce more OTA than the parental strain.•The expression of mfsA is up regulated by hydrogen peroxide.A. Crespo-Sempere was recipient of a FPI fellowship from the Spanish Government. This research was supported by grants AGL2005-00707 and AGL2008-04828-C03-02 from the Spanish Government.Peer Reviewe

Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes

An enhanced green fluorescent protein has been used to tag an OTA-producing strain of Aspergillus carbonarius (W04-40) isolated from naturally infected grape berries. Transformation of the fungus was mediated by Agrobacterium tumefaciens. The most efficient transformation occurred when the co-cultivation was done with 104 conidia due to higher frequency of resistance colonies (894 per 104 conidia) and lower background obtained. To confirm the presence of the hph gene in hygromycin resistant colonies, 20 putative transformants were screened by PCR analysis. The hph gene was identified in all the transformants. Variation on the expression levels of the eGFP was detected among the transformants and 50% of them appeared bright green fluorescent under the microscope. Microscopic analysis of all the bright fluorescent transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the conidia. eGFP expression in A. carbonarius was shown to be stable in all transformants. Confocal Laser scanning microscopy images of grape berries infected with the eGFP transformant demonstrated fungal penetration into the berry tissues. OTA production was importantly increased in the eGFP transformant in comparison with the wild type strain and pathogenicity on grape berries was slightly decreased after four days of inoculation. However, no differences in virulence were found after seven days of inoculation, thus allowing utilization of this eGFP mutant for in situ analysis of A. carbonarius infection of grape berries. To our knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring Aspergillus rot on grape berries.This research was supported by AGL2005-00707/ALI and AGL2008-04828-C03-02 grants from the Spanish Government.Peer Reviewe

Characterization and disruption of the cipC gene in the ochratoxigenic fungus Aspergillus carbonarius

Aspergillus carbonarius is considered the most important ochratoxin A (OTA) producing fungi among those causing OTA contamination in grapes and grape-derived products. CipC is a small protein with unknown function that was previously found to be highly up-regulated in an OTA producer strain of A. carbonarius in comparison to a non OTA producer strain. In this study, cipC was characterized and disrupted via Agrobacterium tumefaciens-mediated transformation in an ochratoxigenic A. carbonarius strain in order to study whether this gene has a role in OTA production. Sequence analysis indicated that the promoter region of cipC contains putative binding sites for transcription factors that regulate the utilization of nutrients, the stress response and detoxification processes, all factors that can influence mycotoxin biosynthesis. Although the {increment}. cipC mutant grew similarly to the wild type strain, the null mutant showed a much higher OTA production. Moreover, when A. carbonarius was grown under the oxidative stress conditions imposed by the presence of hydrogen peroxide, cipC gene expression was up-regulated. These results indicate that cipC is not directly involved in OTA biosynthesis, but sequence analysis of the A. carbonarius cipC gene promoter and the phenotype of the δ cipC disrupted mutant suggests that CipC could be a stress response protein that would be up-regulated concomitantly with OTA production. © 2013 Elsevier Ltd.A. Crespo-Sempere was recipient of a FPI fellowship from the Spanish Government. This research was supported by grants AGL2005-00707 and AGL2008-04828-C03-02 from the Spanish Government.Peer Reviewe

Antifungal Peptides and Proteins to Control Toxigenic Fungi and Mycotoxin Biosynthesis

The global challenge to prevent fungal spoilage and mycotoxin contamination on food and feed requires the development of new antifungal strategies. Antimicrobial peptides and proteins (AMPs) with antifungal activity are gaining much interest as natural antifungal compounds due to their properties such as structure diversity and function, antifungal spectrum, mechanism of action, high stability and the availability of biotechnological production methods. Given their multistep mode of action, the development of fungal resistance to AMPs is presumed to be slow or delayed compared to conventional fungicides. Interestingly, AMPs also accomplish important biological functions other than antifungal activity, including anti-mycotoxin biosynthesis activity, which opens novel aspects for their future use in agriculture and food industry to fight mycotoxin contamination. AMPs can reach intracellular targets and exert their activity by mechanisms other than membrane permeabilization. The mechanisms through which AMPs affect mycotoxin production are varied and complex, ranging from oxidative stress to specific inhibition of enzymatic components of mycotoxin biosynthetic pathways. This review presents natural and synthetic antifungal AMPs from different origins which are effective against mycotoxin-producing fungi, and aims at summarizing current knowledge concerning their additional effects on mycotoxin biosynthesis. Antifungal AMPs properties and mechanisms of action are also discussed

Real-time PCR based procedures for detection and quantification of Aspergillus carbonarius in wine grapes

9 pages, 4 tables, 1 figure.Aspergillus carbonarius is the main species responsible for ochratoxin A accumulation in wine grapes and consequently, its rapid and sensitive detection is increasingly investigated. A new real-time PCR (RTi-PCR) based procedure was developed for the rapid and specific detection and quantification of A. carbonarius in wine grapes. The procedure includes the use of the pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors, and DNA extraction with the EZNA Fungal DNA kit. It reduced the time for A. carbonarius DNA extraction from grapes to 30 min. Two specific primers (AcKS10L/AcKS10R) delimiting a 161 bp fragment, and a probe were designed and directed to the β-ketosynthase domain of a polyketide synthase from A. carbonarius. Specificity was confirmed by testing primers towards purified DNA from 52 fungal strains, including reference and food isolates. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated conidial suspensions from A. carbonarius. The SYBR-Green I and TaqMan RTi-PCR approaches established were able to detect at least 2.4 and 24 genomic equivalents, respectively, using purified DNA. Results obtained from conidial suspensions, after DNA extraction, showed that at least 5 conidia per reaction should be present for a positive result with SYBR-Green I and 50 in the case of TaqMan. The quantification of fungal genomic DNA in artificially inoculated wine grapes performed successfully, with a minimum threshold of 103 conidia mL− 1 for accurate quantification. The developed RTi-PCR assay is a promising tool in the prediction of potential ochratoxigenic risk, even in the case of low-level infections, and suitable for a rapid, automated and high throughput analysis.The authors are grateful to Spanish CICYT (Comisión Interministerial de Ciencia y Tecnología) projects AGL2005-00707 and AGL2004-03060 for financial support. M.V.S. is grateful to the CSIC (Spanish National Research Council) for an I3P contract (I3PDR-7-01). P.V.M.C. is supported by a Ramón y Cajal research contract, cofinanced by the Spanish Government and the University of Valencia.Peer reviewe

Genes differentially expressed by Aspergillus carbonarius strains under ochratoxin A producing conditions

Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus that is responsible for toxin contamination of grapes and wine, coffee and cocoa. A suppression subtractive hybridization (SSH) approach was performed with two strains of A. carbonarius, antagonistic in their OTA-production ability, to identify genes whose expression is linked with the ability to produce OTA. BlastX analysis identified 109 differentially-expressed sequences putatively involved in the production of OTA, with significant similarities (Evalue < 10− 5) to sequences deposited in the NCBI non-redundant protein database. Of the 109 ESTs, 26% were involved in regulation processes, 15% corresponded to hypothetical proteins, 12% were involved in stress response and detoxification, 9% corresponded to transport and secretion processes, 7% corresponded to amino acid metabolism, 7% were involved in hydrolysis of energy reserves and 5% involved in secondary metabolism. Other unisequences showed homology to genes involved in protein synthesis and general metabolism. According to their sequence similarities to genes in the NCBI database, the possible functional roles they might play in the production and regulation of OTA are discussed. Worth noting is the high percentage of genes involved in regulation, including specific and global regulators. It is also important to note the high percentage of genes involved in the response to stress and detoxification.This research was supported by grants from the Generalitat Valenciana (GV2004-B-195) and the Spanish Government (AGL2005-00707/ALI)Peer reviewe

Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction

9 pages.This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the β-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic Tm-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC®-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.Peer reviewe

Antifungal peptides and proteins to control toxigenic fungi and mycotoxin biosynthesis

The global challenge to prevent fungal spoilage and mycotoxin contamination on food and feed requires the development of new antifungal strategies. Antimicrobial peptides and proteins (AMPs) with antifungal activity are gaining much interest as natural antifungal compounds due to their properties such as structure diversity and function, antifungal spectrum, mechanism of action, high stability and the availability of biotechnological production methods. Given their multistep mode of action, the development of fungal resistance to AMPs is presumed to be slow or delayed compared to conventional fungicides. Interestingly, AMPs also accomplish important biological functions other than antifungal activity, including anti-mycotoxin biosynthesis activity, which opens novel aspects for their future use in agriculture and food industry to fight mycotoxin contamination. AMPs can reach intracellular targets and exert their activity by mechanisms other than membrane permeabilization. The mechanisms through which AMPs affect mycotoxin production are varied and complex, ranging from oxidative stress to specific inhibition of enzymatic components of mycotoxin biosynthetic pathways. This review presents natural and synthetic antifungal AMPs from different origins which are effective against mycotoxin-producing fungi, and aims at summarizing current knowledge concerning their additional effects on mycotoxin biosynthesis. Antifungal AMPs properties and mechanisms of action are also discussed.This work was supported by grant RTI2018-101115B-C21 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”, PROMETEO/2018/066 from ‘Conselleria d’Educació’ (Generalitat Valenciana, Comunitat Valenciana, Spain).Peer reviewe

Proteomic Analysis of Saccharomyces cerevisiae Response to Oxidative Stress Mediated by Cocoa Polyphenols Extract

The present study addressed the protective effects against oxidative stress (OS) of a cocoa powder extract (CPEX) on the protein expression profile of S. cerevisiae. A proteomic analysis was performed after culture preincubation with CPEX either without stress (−OS) or under stress conditions (+OS) (5 mM of H2O2). LC-MS/MS identified 33 differentially expressed proteins (–OS: 14, +OS: 19) that were included By Gene Ontology analysis in biological processes: biosynthesis of amino acids, carbohydrate metabolism and reactive oxygen species metabolic process. In a gene-knockout strains study, eight proteins were identified as putative candidates for being involved in the protective mechanism of cocoa polyphenols against OS induced by H2O2. CPEX was able to exert its antioxidant activity in yeast mainly through the regulation of: (a) amino acids metabolism proteins by modulating the production of molecules with known antioxidant roles; (b) stress-responsive protein Yhb1, but we were unable to fully understand its down-regulation; (c) protein Prb1, which can act by clipping Histone H3 N-terminal tails that are related to cellular resistance to DNA damaging agents.This work was supported by the Spanish Ministry of Science and Innovation (MICINN) (Project AGL2008-02208/ALI)Peer reviewe

Development of a FungalBraid Penicillium expansum-based expression system for the production of antifungal proteins in fungal biofactories

Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost-effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum-based expression system that consisted of the paf gene promoter, signal peptide (SP)-pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.This work was supported by grant RTI2018-101115B-C21 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”, PROMETEO/2018/066 from ‘Conselleria d’Educació’ (Generalitat Valenciana, Comunitat Valenciana, Spain) and by grant PCI2018-092893 funded by MCIN/AEI/ 10.13039/501100011033 and by “The European Union” (SUSPHIRE Project, ERACoBioTech; Horizon 2020 grant agreement No. 722361). EM and MGL were recipients of predoctoral grants FPU18/02019 and FPU19/02066 funded by MCIN/AEI/ 10.13039/501100011033 and by “ESF Investing in your future”.Peer reviewe