Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response

Highlights

• Piperine inhibits AFB1 production by transcriptional regulation of cluster genes.

• Piperine inhibition of AFB1 production goes with an enhancement of antioxidant status in A. flavus.

• Over-expression of bZIP-type factors are involved in AFB1 inhibition by piperine.

Abstract

Aspergillus flavus, a soil-borne pathogen, represents a danger for humans and animals since it produces the carcinogenic mycotoxin Aflatoxin B1 (AFB1). Approaches aiming the reduction of this fungal contaminant mainly involve chemicals that may also be toxic. Therefore, identification and characterization of natural anti-aflatoxigenic products represents a sustainable alternative strategy. Piperine, a major component of black and long peppers, has been previously demonstrated as an AFB1-inhibitor; nevertheless its mechanism of action was yet to be elucidated. The aim of the present study was to evaluate piperine’s molecular mechanism of action in A. flavus with a special focus on oxidative stress response. For that, the entire AFB1 gene cluster as well as a targeted gene-network coding for fungal stress response factors and cellular receptors were analyzed. In addition to this, fungal enzymatic activities were also characterized. We demonstrated that piperine inhibits aflatoxin production and fungal growth in a dose-dependent manner. Analysis of the gene cluster demonstrated that almost all genes participating in aflatoxin’s biosynthetic pathway were down regulated. Exposure to piperine also resulted in decreased transcript levels of the global regulator veA together with an over-expression of genes coding for several basic leucine zipper (bZIP) transcription factors such as atfA, atfB and ap-1 and genes belonging to superoxide dismutase and catalase’s families. Furthermore, this gene response was accompanied by a significant enhancement of catalase enzymatic activity. In conclusion, these data demonstrated that piperine inhibits AFB1 production while positively modulating fungal antioxidant status in A. flavus.

Introduction

Mycotoxins are secondary metabolites produced by some filamentous fungi. Among the 300 reported mycotoxins, AFB1 is the most dangerous one. In fact, AFB1 is a potent carcinogenic agent in humans inducing liver cancer (IARC, 1993, Wu et al., 2014). This mycotoxin is synthesized by at least 15 species belonging to the Flavi section of the Aspergillus genus, A. flavus considered as the most frequent source of aflatoxins in crops (Varga et al., 2015). To date, it is estimated that at least 500 million of people living in sub-Saharan Africa, Latin America and Asia, are exposed to increasing levels of mycotoxins, including aflatoxins, through contaminated commodities (Pitt et al., 2012). In 2010, analysis of several foodborne chemicals by the Chemical and Toxins Disease Task Force reported that aflatoxin was associated with the highest number of DALYs (deaths and disability adjusted life years) (636, 869) where one DALY represents the sum of years lived with disability and years of life lost (Gibb et al., 2015). Worryingly, surveys demonstrated that higher levels of aflatoxins in agricultural commodities were linked to upraising climatic conditions (Streit et al., 2013). Indeed, AFB1’s contamination was recently reported on crops from other temperate regions of Europe, demonstrating an increased distribution of this mycotoxin potentially related to global climate changes (Battilani et al., 2016, Perrone et al., 2014). Even if several approaches have been developed to limit AFB1’s contamination (Wild et al., 2015, Jalili, 2015), they remain insufficient to completely avoid mycotoxin contamination thus emphasizing the need to develop new strategies to guarantee food safety. The use of natural products such as spices, plant extracts or essential oils with anti-aflatoxinogenic properties represent a complementary or alternative strategy against mycotoxin contamination (Annis et al., 2000, El Khoury et al., 2017, Holmes et al., 2008, Sakuda et al., 2016). For instance, piperine, a major active component of black and long peppers (Piper nigrum L. and Piper longum L.) has been previously demonstrated as an effective inhibitor of aflatoxin production (Lee et al., 2002, Madhyastha and Bhat, 1984). However, the molecular mechanism of action by which this molecule inhibits AFB1 production has not been studied. In the last years, aflatoxin biosynthesis has been closely linked to fungal oxidative stress status (Montibus et al., 2013, Roze et al., 2013, Amare and Keller, 2014). Indeed, studies in Aspergilli demonstrated several molecules with antioxidant capacities are able to inhibit aflatoxin production (Jahanshiri et al., 2012, Liang et al., 2015). Furthermore, it has been demonstrated that a regulatory network of genes coding for stress response transcription factors (AtfB, SrrA, Ap-1 and MsnA) is directly involved in aflatoxin biosynthesis (Hong et al., 2013). Within this context, the present study aimed to elucidate the anti-aflatoxinogenic mechanism of action of piperine by studying its impact on the expression of 27 genes involved in AFB1 gene cluster and 20 genes coding for oxidative stress response in A. flavus, by using a large-scale q-PCR approach (Caceres et al., 2016).

We demonstrated that piperine resulted in a down-regulation of almost all genes of the AFB1 cluster leading to the subsequent inhibition of the mycotoxin biosynthesis. We also showed that exposure to piperine induced an inhibition by 50% of veA expression levels together with an over-expression of several bZIP transcription factors (atfA, atfB and ap-1) and genes coding for antioxidant defense belonging to catalase and superoxide dismutase families (sod1, catA and cat2). Simultaneously, a significant increase in catalase enzymatic activity was also observed.