Molecular mechanisms of Aspergillus flavus secondary metabolism and development

Highlights

• Eight secondary metabolite gene clusters have been characterized in Aspergillus flavus, a pathogen of both plants and humans.

• Aspergillus flavus contains a quorum sensing system regulating development and secondary metabolism.

• bZIP mediated pathways regulate stress responses in Aspergillus flavus.

Abstract

The plant and human opportunistic fungus Aspergillus flavus is recognized for the production of the carcinogen aflatoxin. Although many reviews focus on the wealth of information known about aflatoxin biosynthesis, few articles describe other genes and molecules important for A. flavus development or secondary metabolism. Here we compile the most recent work on A. flavus secondary metabolite clusters, environmental response mechanisms (stress response pathways, quorum sensing and G protein signaling pathways) and the function of the transcriptional regulatory unit known as the Velvet Complex. A comparison to other Aspergilli reveals conservation in several pathways affecting fungal development and metabolism.

Introduction

Aspergillus flavus is a ubiquitous saprophytic fungus found in soils across the world. Although first described in 1809, A. flavus was thrown into the limelight in 1962 as a result of the Turkey X disease that killed thousands of poultry (Nesbitt et al., 1962). The Turkey X outbreak led to the discovery of aflatoxin, a fungal mycotoxin that had contaminated the poultry feed. Since then, A. flavus and aflatoxin have had tremendous economic and health impacts across the world (Amaike and Keller, 2011). Although aflatoxin is noted as the primary metabolite causing human disease, the fungus produces several toxic metabolites that may also contribute to ill health as covered below.

A. flavus survives as conidia or sclerotia in soil and organic debris. While conidia allow the fungus to mass-disseminate, sclerotia enable survival in harsh environmental conditions and can germinate once conditions improve. On host tissue, including that of humans, animals and plants, conidia germinate and grow as mycelia which can develop into either conidiophores or sclerotia depending on environmental and nutritional cues. While the conidium is considered the predominant infectious spore, the predominant reproductive form in soil is not known. Sclerotia contain the sexual ascospores of the fungus, which until recently were only reported to occur in experimental laboratory conditions but have now been reported to occur in the field also (Horn et al., 2009, Horn et al., 2013).

A. flavus colonizes and produces aflatoxins (there are several forms of aflatoxin with aflatoxin B1 being the most carcinogenic) in oil-rich agricultural crops including maize, peanuts and cottonseeds both pre- and post-harvest. As aflatoxin is toxigenic as well as carcinogenic, controlling aflatoxin contamination of crops is vital. However, controlling contamination, both pre- and post-harvest, induces tremendous monetary losses worldwide. In the US, aflatoxin contamination incurs economic losses of approximately US $1 billion per annum (Vardon et al., 2003) while African countries lose approximately $670 million from failure to meet European export standards (Otsuki et al., 2001). In an effort to curb aflatoxin exposure, the US Food and Drug Administration only allows 20 ppb in food and 0.5 ppb in milk (Georgianna and Payne, 2009) while some countries such as those in Europe have even stricter guidelines. On the other hand, developing countries often have lax, if any, guidelines for aflatoxin contamination.

Aflatoxins have a wide range of health impacts depending on the aflatoxin dose. Acute aflatoxicosis, arising from high-dose aflatoxin intake over a short period, often results in aflatoxin-poisoning outbreaks killing scores of people. The quintessential examples for this are the recurrent outbreaks seen in the East African country Kenya, which experienced its worst outbreak in 2004 with 317 cases and 125 reported deaths (Azziz-Baumgartner et al., 2005). Chronic aflatoxicosis, arising from low-dose aflatoxin consumption over an extended period, can result in immune suppression, stunting and liver cancer. Aflatoxin-induced liver cancer is known to arise from a mutation in the tumor suppressor gene, p53, in the liver (Hsu et al., 1991). Perhaps exacerbating the problem is the fact that exposure to aflatoxin B1 in hepatitis B virus-endemic areas highly increases the chances of developing hepatocellular carcinoma by as much as 30-fold, creating severe health problems in developing countries where both are common occurrences (Groopman et al., 2008). To a lesser extent, aflatoxin B1 and hepatitis C virus also exhibit a similar relationship leading to increased chances of developing hepatocellular carcinoma (Kuang et al., 2005).

A. flavus also causes mycoses (infection with fungus as opposed to diseases caused by consumption of fungal toxins which are broadly known as mycotoxicoses) in humans and animals. A. flavus is unique in that it is an opportunistic pathogen of both plants and animals (Gauthier and Keller, 2013, Hedayati et al., 2007). In humans, A. flavus is the second most common cause of invasive aspergillosis accounting for 10–20% of infections; only second to A. fumigatus which accounts for 80–90% of invasive aspergillosis infections (Krishnan et al., 2009). In hot and dry areas like Africa and the Middle East, A. flavus causes the majority of cases of fungal sinusitis, keratitis and cutaneous infections (Khairallah et al., 1992, Krishnan et al., 2009). Animals including rabbits, chickens and turkey are also highly susceptible to aspergillosis from A. flavus infection.

In this review, we will highlight genes and molecules important in secondary metabolism and development of A. flavus and related species. The reader is also referred to other reviews for greater coverage of specific areas of research on this fungus and/or aflatoxin biosynthesis (Chang and Ehrlich, 2013, Khlangwiset et al., 2011, Woloshuk and Shim, 2013) as well as on environmental factors that influence host-pathogen interaction between A. flavus and maize (Fountain et al., 2013).