Review

# Immunomodulatory Effects of Edible and Medicinal Mushrooms and Their Bioactive Immunoregulatory Products

Shuang Zhao <fnref n="1" />, Qi Gao <fnref n="1" />, Chengbo Rong <fnref n="1" />, Shouxian Wang <fnref n="1" />, Zhekun Zhao <fnref n="1" />,<fnref n="2" />, Yu Liu <fnref n="1" /> and Jianping Xu <fnref n="3" />,*

Received: 10 October 2020; Accepted: 2 November 2020; Published: 8 November 2020

<figure><img src="image_144.png" /></figure>

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**Abstract:** Mushrooms have been valued as food and health supplements by humans for centuries. They are rich in dietary fiber, essential amino acids, minerals, and many bioactive compounds, especially those related to human immune system functions. Mushrooms contain diverse immunoregulatory compounds such as terpenes and terpenoids, lectins, fungal immunomodulatory proteins (FIPs) and polysaccharides. The distributions of these compounds differ among mushroom species and their potent immune modulation activities vary depending on their core structures and fraction composition chemical modifications. Here we review the current status of clinical studies on immunomodulatory activities of mushrooms and mushroom products. The potential mechanisms for their activities both in vitro and in vivo were summarized. We describe the approaches that have been used in the development and application of bioactive compounds extracted from mushrooms. These developments have led to the commercialization of a large number of mushroom products. Finally, we discuss the problems in pharmacological applications of mushrooms and mushroom products and highlight a few areas that should be improved before immunomodulatory compounds from mushrooms can be widely used as therapeutic agents.

**Keywords:** bioactive compounds; FIP; human health; immunomodulation; induced apoptosis; lectin; medicinal mushrooms; polysaccharide; terpenes and terpenoids

## 1. Introduction

In clinical practice, immunomodulators are usually classified into three categories: immunosuppressants, immunostimulants, and immunoadjuvants [1]. Their market share has increased rapidly over the past few years due to wide-ranging medical applications for patients that require human immune system modulations. Immune system modulations are also commonly used as prophylactic medicine for an increasing number of healthy people [2,3]. While most immunomodulators are synthetic or semi-synthetic compounds, there has been a growing interest in natural immunomodulators. Many natural compounds have shown significant immunomodulatory and overall health-benefiting effects to humans, with no or minimal toxicity. These natural-based products with potential pharmacological and beneficial effects are increasingly perceived as safer than synthetic compounds by the general public [4,5]. Indeed, many of the currently used chemical drugs have negative side effects

<footnote n="1">¹ Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China; zhaoshuang@ipepbaafs.cn (S.Z.); gaoqi@ipepbaafs.cn (Q.G.); rongchengbo@ipepbaafs.cn (C.R.); wangshouxian@ipepbaafs.cn (S.W.); zhaozhekun@ipepbaafs.cn (Z.Z.); liuyu@ipepbaafs.cn (Y.L.)</footnote>

<footnote n="2">² College of Life Sciences and Food Engineering, Hebei University of Engineering, Handan 056038, China</footnote>

<footnote n="3">³ Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada</footnote>

<footnote>* Correspondence: jpxu@mcmaster.ca</footnote>
and the market share of natural immunomodulators is increasing rapidly with an annual growth rate of 8.6% [1,6].

Medicinal mushrooms (MMs) are an important source of natural immunomodulators. Representing a subset of all mushrooms, MMs are broadly defined as macroscopic fungi that are used in the form of extracts or whole mushroom powder for human health benefits [7]. The health benefits may come in the form of helping to prevent and/or treat diseases in humans, and to create a dietary balance of a healthy diet. Dating back to thousands of years, MMs have been historically used as herbal medicines for human health, such as for the treatment of infectious diseases, gastrointestinal disorders and asthmatic conditions [8]. The biomass or specific extracts from all developmental stages of MMs, including the fruiting body, sclerotium, mycelium, and spores, have been used as health foods or dietary supplements [9,10]. Some of the extracted nutrients from mushrooms known as mushroom nutraceuticals have been made into capsules or tablets as dietary supplements. Regular intake of these nutraceuticals has been associated with enhancement of the human immune response, leading to increased resistance to infectious diseases and faster recovery from a diversity of diseases [11].

At present, thousands of branded MM products are sold all over the world. The health benefits of MM products include anticancer, immune-stimulation, antioxidant, antihyperglycemic, antihypertensive, neuroprotective, hepatoprotective, antidiabetic, antifungal, antibacterial, and antiviral activities [7,12]. Their effects have been attributed to many components, such as minerals, essential amino acids, dietary fiber, proteins, polysaccharides, lipopolysaccharides, glycoproteins, and secondary metabolites. Among these, some of the complex organic compounds have shown immunomodulatory effects [7]. For example, polysaccharides from MMs can activate natural killer cells, macrophages, and neutrophils, as well as induce innate immune cytokines and interleukins. In addition, secondary metabolites from MMs, such as sterols, terpenes, and phenols can enhance the survival of hosts by stabilizing their important metabolic functions [8].

Different MMs contain different functional components that may impact the same or different immunomodulatory pathways at varying efficacies. In the following sections, we first provide a brief description of the known MMs. We then summarize the diversity, structure, function, and molecular mechanism of action of functional ingredients from MMs that have shown to be involved in immunomodulation. We finish by briefly describing how genomics can accelerate research on medicinal mushrooms.

## 2. Medicinal Mushrooms

As mentioned above, medicinal mushrooms refer to all macroscopic fungi whose extracts or powder form from any stages of the mushroom development have shown documented beneficial effects on health [13]. These beneficial effects may have been shown in the forms of in vitro, ex vivo, or in vivo activities. Their effects may cover different groups of organisms such as antagonistic effects against human pathogens and parasites, and/or beneficial effects for human and animal cell lines, or animal and human individuals [11]. Since many edible mushrooms and their products have shown to be a beneficial component of the human diet, some of these edible mushrooms are also commonly included as medicinal mushrooms [7]. In our literature search, a large number of MMs have been documented. For example, terpenes and terpenoids from *Ganoderma lucidum* could stimulate the expressions of genes coding for proteins in the nuclear factor (NF)-κB pathway and modulate immune system functions [14]. Heteroglycan and heteroglycan-peptide from the mushroom of *Hericium erinaceus* can modulate the immuno-effects by inducing nitric oxide production and increasing expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-12 [15]. These mushrooms belong to two fungal phyla, Ascomycota and Basidiomycota. Most of the MMs are in phylum Basidiomycota. Table 1 shows the major medicinal mushrooms, including their taxonomy and geographic/ecological distributions. As can be seen, some of these mushrooms are broadly distributed (e.g., the button mushroom *Agaricus bisporus*) while others are geographically more restricted (e.g., the Himalayan caterpillar fungus *Ophiocyceps sinensis*). Some of the mushrooms included in the table e.g., *Amanita phalloides* are highly poisonous when
consumed by humans. However, the dilutions of an *A. phalloides* extract that contains the toxin amanitin have shown to be effective as an anti-tumor therapy [16].

<table><caption>**Table 1.** Major medicinal mushrooms and their main distributions.</caption><thead><tr><td>MM Species</td><td>Common Name</td><td>Taxonomy</td><td>Geographic/Ecological Distribution</td></tr></thead><tbody><tr><td>Agrocybe aegerita</td><td>Black Poplar mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Bolbitiaceae</td><td>North temperate and<br>subtropical zone</td></tr><tr><td>Agaricus bisporus</td><td>Button mushroom,<br>Portobello mushroom,<br>Common mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Agaricaceae</td><td>USA, China, France, Netherlands,<br>United Kingdom, Italy, Poland,<br>Spain, Germany, Canada, Ireland,<br>Belgium, Indonesia, Hungary and<br>Mexico</td></tr><tr><td>Agaricus blazei<br>(syn. Agaricus<br>brasiliensis)</td><td>Royal Sun Agaricus,<br>Almond Portobello</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Agaricaceae</td><td>America, Brasil, Japan, China</td></tr><tr><td>Amanita phalloides</td><td>Death Cap</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Amanitaceae</td><td>Europe, North American, Asia</td></tr><tr><td>Boletus edulis</td><td>Cep, Porcini, Penny<br>Bun Bolete</td><td>Basidiomycota<br>Agaricomycetes<br>Boletales<br>Boletaceae</td><td>China, Italy, France, Swiss,<br>Germany</td></tr><tr><td>Boletus speciosus</td><td>Red-Capped Butter<br>Bolete</td><td>Basidiomycota<br>Agaricomycetes<br>Boletales<br>Boletaceae</td><td>Eastern North America,<br>Southwest of China and Europe</td></tr><tr><td>Chroogomphus rutilus</td><td>Copper Spike</td><td>Basidiomycota<br>Agaricomycetes<br>Boletales<br>Gomphidiaceae</td><td>China</td></tr><tr><td>Clitocybe nebularis</td><td>Clouded Funnel</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Tricholomataceae</td><td>China, Japan, Taiwan, Europe,<br>North America, North Africa</td></tr><tr><td>Cryptoporus volvatus</td><td>Veiled Polypore</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Trunks of pine, fir and spruce</td></tr><tr><td>Dichomitus squalens</td><td>Common White-Rot<br>fungus</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Trunks of conifers such as pine<br>and larch</td></tr><tr><td>Flammulina velutipes</td><td>Golden Needle<br>mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Physalacriaceae</td><td>Subtropical zone such as Japan,<br>Russia, Australia and other<br>countries as well as Europe,<br>North America</td></tr><tr><td>Floccularia luteovirens<br>(syn. Armillaria<br>luteovirens)</td><td>Scaly Yellow<br>mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Tricholomataceae</td><td>Meadow at altitudes of<br>3000–4000 m above sea level</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>MM Species</th><th>Common Name</th><th>Taxonomy</th><th>Geographic/Ecological Distribution</th></tr></thead><tbody><tr><td>Ganoderma atrum</td><td>Black Ling-zhi</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Tropical regions</td></tr><tr><td>Ganoderma capense</td><td>Dark Ling-zhi</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Tropical regions</td></tr><tr><td>Ganoderma japonicum</td><td>Bloody Ling-zhi</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Majority in tropical and<br>subtropical regions of Asia,<br>Australia, Africa and America,<br>minority in temperate zone</td></tr><tr><td>Ganoderma lucidum</td><td>Reitake,<br>Ling-zhi,<br>Spirit Plant<br>Reishi</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Majority in tropical and<br>subtropical regions of Asia,<br>Australia, Africa and America,<br>minority in temperate zone</td></tr><tr><td>Ganoderma microsporum</td><td>Small-Spored Ling-zhi</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Subtropics zone</td></tr><tr><td>Ganoderma lingzhi</td><td>Ling-zhi</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>China, North Korea, Japan</td></tr><tr><td>Ganoderma sinensis</td><td>Zi-zhi</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>China, North Korea, Japan</td></tr><tr><td>Ganoderma tsugae</td><td>Hemlock Varnish Shelf</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Northern and Montaine zone</td></tr><tr><td>Grifola frondosa</td><td>Maitake<br>Hen of the Woods</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Grifolaceae</td><td>Japan, China</td></tr><tr><td>Hericium erinaceus</td><td>Lion's Mane<br>mushroom, Bearded<br>Tooth mushroom,<br>Monkey-Head<br>mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Russulales<br>Hericiaceae</td><td>Broad-leaved forest or coniferous<br>and broad-leaved mixed forest in<br>northern temperate zone such as<br>Western Europe, North America,<br>China, Japan, Russia</td></tr><tr><td>Inonotus obliquus</td><td>Clinker Polypore, Birch<br>Conk, Chaga</td><td>Basidiomycota<br>Agaricomycetes<br>Hymenochaetacles<br>Hymenochaetaceae</td><td>Russia, China</td></tr><tr><td>Lentinula edodes</td><td>Shiitake, Black Forest<br>mushroom, Golden<br>Oak mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Omphalotaceae</td><td>Distributed in an arc area on the<br>west side of the Pacific Ocean,<br>Japan, Papua New Guinea, Nepal,<br>the Mediterranean coast and<br>northern Africa</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>MM Species</th><th>Common Name</th><th>Taxonomy</th><th>Geographic/Ecological Distribution</th></tr></thead><tbody><tr><td>Lignosus rhinocerotis</td><td>Tiger Milk mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>China, Indonesia, Philippines,<br>Sri Lanka, Australia, Thailand,<br>Malaysia, Papua New Guinea and<br>rainforests of East Africa</td></tr><tr><td>Leucocalocybe<br>mongolica<br>(syn. Tricholoma<br>mongolicum)</td><td>Mongolia mushroom</td><td>Basidiomycota<br>Basidiomycetes<br>Agaricales<br>Agaricales incertae sedis</td><td>Inner Mongolia in China</td></tr><tr><td>Marasmius oreades</td><td>Fairy Ring mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Marasmiaceae</td><td>North America and Asia</td></tr><tr><td>Morchella esculenta</td><td>Common Morel,<br>Yellow Morel, Sponge<br>Morel</td><td>Ascomycota<br>Pezizomycetes<br>Pezizales<br>Morohellaceae</td><td>Widely cultured over the world<br>such as France, Germany,<br>America, India, China, Russia,<br>Sweden, Mexico, Spain,<br>Czechoslovakia and Pakistan</td></tr><tr><td>Morchella conica</td><td>Black Morel, Sponge<br>mushroom</td><td>Ascomycota<br>Pezizomycetes<br>Pezizales<br>Morohellaceae</td><td>Distributed under broad-leaved<br>forest, coniferous broad-leaved<br>mixed forest, forest edge open<br>space and weeds</td></tr><tr><td>Naematelia aurantialba<br>(syn. Tremella<br>aurantialba)</td><td>Golden Tremella</td><td>Basidiomycota<br>Tremellomycetes<br>Tremellales<br>Naemateliaceae</td><td>Mountain forest of quercus,<br>mutualism with Stereum spp.</td></tr><tr><td>Ophiocordyceps<br>sinensis</td><td>Caterpillar fungus,<br>Himalaya Viagra</td><td>Ascomycota<br>Sordariomycetes<br>Hypocreales<br>Ophiocordycipitaceae</td><td>Southwest China, Nepal</td></tr><tr><td>Pholiota adiposa</td><td>Chestnut mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Strophariaceae</td><td>Distributed on the dead willows<br>in the forest in China</td></tr><tr><td>Pleurotus<br>citrinopileatus</td><td>Golden Oyster<br>mushroom,<br>Tamogitake</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Pleurotaceae</td><td>Widely cultured all over the world</td></tr><tr><td>Pleurotus ostreatus</td><td>Oyster mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Pleurotaceae</td><td>Widely cultured all over the world</td></tr><tr><td>Cerioporus squamosus<br>(syn. Polyporus<br>squamosus)</td><td>Dryad's Saddle,<br>Pheasant's Back<br>mushroom</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Widely distributed in hardwood<br>forest of North America, Australia,<br>Asia and Europe</td></tr><tr><td>Poria cocos</td><td>Fuling, China Root</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Laetiporaceae</td><td>Parasitic on the roots of Pinaceae<br>plants, mainly distributed in<br>China</td></tr><tr><td>Rhodonia placenta<br>(syn. Postia placenta)</td><td>Rosy Crust</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Dacryobolaceae</td><td>Widely distributed all over the<br>world</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>MM Species</th><th>Common Name</th><th>Taxonomy</th><th>Geographic/Ecological Distribution</th></tr></thead><tbody><tr><td>Pseudosperma<br>umbrinellum<br>(syn. Inocybe<br>umbrinella)</td><td>Fibrous Hat</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Inocybaceae</td><td>France</td></tr><tr><td>Russula delica</td><td>Milk-White Brittle-gill</td><td>Basidiomycota<br>Agaricomycetes<br>Russulales<br>Russulaceae</td><td>Taiga forest and mixed forests</td></tr><tr><td>Russula lepida</td><td>Rosy Russula</td><td>Basidiomycota<br>Agaricomycetes<br>Russulales<br>Russulaceae</td><td>Widely distributed all over the<br>world</td></tr><tr><td>Sarcodon aspratus</td><td>Black Tiger Paw</td><td>Basidiomycota<br>Agaricomycetes<br>Thelephorales<br>Thelephoraceae</td><td>Southwest of China</td></tr><tr><td>Schizophyllum<br>commune</td><td>Split Gill</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Schizophyllaceae</td><td>Widely distributed all over the<br>world</td></tr><tr><td>Stropharia<br>rugosoannulata</td><td>Wine Cap Stropharia,<br>Garden Giant,<br>Burgundy mushroom,<br>King Stropharia</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Strophariaceae</td><td>Europe, North America, Asia</td></tr><tr><td>Taiwanofungus<br>camphoratus<br>(syn. Antrodia<br>camphorate)</td><td>Poroid Brown-rot<br>fungus, Stout Camphor<br>fungus</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales incertae sedis</td><td>Mountain forest in Taiwan with<br>altitudes of 450–2000 m</td></tr><tr><td>Trametes versicolor<br>(syn. Polystictus<br>versicolor)</td><td>Turkey Tail fungus</td><td>Basidiomycota<br>Agaricomycetes<br>Polyporales<br>Polyporaceae</td><td>Global distribution; Broad-leaf<br>woods</td></tr><tr><td>Tropicoporus linteus<br>(syn. Phellinus<br>linteus)</td><td>Mesima, Black Hoof<br>fungus</td><td>Basidiomycota<br>Agaricomycetes<br>Hymenochaetales<br>Hymenochaetaceae</td><td>Distributed on the dead trees and<br>trunks in China</td></tr><tr><td>Xerocomellus<br>chrysenteron<br>(syn. Xerocomus<br>Chrysenteron)</td><td>Red Cracking Bolete</td><td>Basidiomycota<br>Agaricomycetes<br>Agaricales<br>Agaricales incertae sedis</td><td>China</td></tr><tr><td>Xylaria hypoxylon</td><td>Candlestick fungus,<br>Candlesnuff fungus,<br>Carbon Antlers, Stag's<br>Horn fungus</td><td>Ascomycota<br>Sordariomycetes<br>Xylariales<br>Xylariaceae</td><td>Northern Europe</td></tr><tr><td>Xylaria nigripes</td><td>Dead Moll's Fingers</td><td>Ascomycota<br>Sordariomycetes<br>Xylariales<br>Xylariaceae</td><td>China, mutualism with white ant</td></tr><tr><td>Volvariella volvacea</td><td>Straw mushroom</td><td>Basidiomycota<br>Basidiomycetes<br>Agaricales<br>Pluteaceae</td><td>China, East Asia, Southeast Asia</td></tr></tbody></table>
Some of these MM species have been used as herbal medicine for centuries, including *Ganoderma lucidum*, *Ganoderma lingzhi*, *Lentinula edodes*, *Inonotus obliquus*, *Fomitopsis officinalis*, *Piptoporus betulinus*, and *Fomes fomentarius* [7,17]. While these mushrooms have attracted most of the medical attention among the MMs, other species in multiple genera have also shown immunomodulatory and anti-tumor effects, such as those in genera *Agaricus*, *Albatrellus*, *Antrodia*, *Calvatia*, *Clitocybe*, *Cordyceps*, *Flammulina*, *Fomes*, *Funlia*, *Ganoderma*, *Inocybe*, *Inonotus*, *Lactarius*, *Phellinus*, *Pleurotus*, *Russula*, *Schizophyllum*, *Suillus*, *Trametes*, and *Xerocomus* [12].

Figure 1 shows a few representative medicinal mushroom species in their natural habitats. Some medicinal mushrooms are only found in the wild, e.g., the ectomycorrhizal mushrooms *Boletus edulis* and *Russula lepida*. However, a large number of medicinal mushrooms are also commercially cultivated, including Shiitake, Ling-zhi, and Lion's Mane. Figure 2 shows a few representative medicinal mushrooms under cultivation.

<figure><img src="image_145.png" /><figcaption>**Figure 1.** A few representative medicinal mushrooms from the wild.</figcaption></figure>
<figure><img src="image_146.png" /><figcaption>**Figure 2.** A few representative cultivated medicinal mushrooms.</figcaption></figure>

**3. Immunomodulatory Compounds and Commercial Products of Medicinal Mushrooms**

As shown above, there is a large number of medicinal mushrooms in diverse taxonomic groups. Some of these medicinal mushrooms are commercially cultivated for food but there is an increasing trend for developing the immune-active compounds from these cultivated mushrooms into nutraceuticals. Tables 2–5 summarize the major groups of bioactive compounds in medicinal mushrooms and their demonstrated immunomodulatory effects to specific pathologies, including the relevant references.

The main classes of compounds from medicinal mushrooms with immunomodulatory properties are terpenes and terpenoids, lectins, fungal immunomodulatory proteins (FIPs), and polysaccharides (particularly β-D-glucans, but also include polysaccharopeptides and polysaccharide proteins) [1]. Below we describe specific examples in each of these groups.

**3.1. Polysaccharides**

Among the bioactive compounds derived from mushrooms with immunomodulatory activity, those based on polysaccharides, with or without side chain modifications (including polysaccharopeptides and polysaccharide proteins) are the most reported during the last several decades [18]. Table 2 presents a list of polysaccharides from medicinal mushrooms that have shown immunomodulatory activities. Among the reported polysaccharides with immunomodulatory and antitumor activities, the best-known is lentinan, isolated from shiitake (L. edodes), as well as schizophyllan from Schizophyllum commune. Both lectinan and schizophyllan contain β-1,3-D-glucans with β-1,6 branches. Specifically, lentinan showed immunomodulatory properties against gastric cancer while schizophyllan was effective against head and neck cancer. Both products have been licensed and approved in Japan since 1986 for clinical use, in combination with chemotherapy against the two respective cancers [19,20].
Other polysaccharide-based compounds showing immunomodulatory properties have a similar core polysaccharide chemical structure but contain different branching linkages and/or branches with different conjugates. These polysaccharide-conjugate complexes are called heteroglucans, with α(1–4)- and β(1–3) glycosidic linkages to protein components. For example, in the presence of fucose (a hexose deoxy sugar with the chemical formula C6H12O5), the turkey tail mushroom *Trametes versicolor* produces a Krestin bound β-glucan polysaccharide K (PSK). PSK is commercially produced from this mushroom in Japan and has been approved for clinical use since 1977 [21]. Several subsequent reports confirmed the effectiveness of PSK as an adjuvant to conventional cancer therapies through inhibition of cancer metastasis [22], induction of cancer cell apoptosis [23], improvement of inflammatory cytokines gene expression [24,25].

Another compound isolated from *T. versicolor* is a polysaccharide peptide (PSP). PSP contains rhamnose and arabinose, two monosaccharides not found in PSK. In addition, the conjugated protein was also different. PSP has been commercially available in the Chinese market since 1987 [21]. It has been documented to improve the quality of life in cancer patients by providing substantial pain relief and enhancing immune status in 70–97% of patients with stomach, esophagus, lung, ovary and cervical cancers. Specifically, PSP has been shown to be capable of boosting immune cell production, ameliorating chemotherapy symptoms, and enhancing tumor infiltration by dendritic and cytotoxic T-cells [26].

Two other well-known polysaccharide-protein complexes produced by *Macrocybe gigantea* (syn. *Tricholoma giganteum*) and *Agaricus brazei*, respectively, have also shown immunomodulatory effects. A polysaccharide-protein complex (PSPC) isolated from *T. giganteum* showed that it could help restore and improve the phagocytic function of macrophages in tumor-bearing mice [13,27]. Similarly, AbM isolated from *A. brazei* contains diverse polysaccharide-protein complexes with different chemical linkages such as β-1,6-glucan, α-1,6- and α-1,4-glucan, glucomannan and β-1,3-glucan. AbM has been shown to have immunomodulatory and antineoplastic properties [28]. Polysaccharides and polysaccharide-protein complexes from other medicinal mushrooms that have shown immunomodulatory effects are listed in Table 2.
<table><caption>**Table 2.** Major immunomodulatory polysaccharides from medicinal mushrooms and their immunomodulatory effects.</caption><thead><tr><td>Source</td><td>Active Compound</td><td>Immunomodulatory Effect</td><td>Refs</td></tr></thead><tbody><tr><td>Agaricus blazei (syn. Agaricus brasiliensis)</td><td>Heteroglycan, Glycoprotein, Glucomannan-protein complex, β-1,3-D-glucan, with β-1,6-D-glucan branch</td><td>Stimulates Natural Killer (NK) cells, macrophages, dendritic cells, and granulocytes; induction of Tumor Necrosis Factor (TNF), Interferon (IFN)-γ, and Interleukin (IL)-8 production</td><td>[29]</td></tr><tr><td>Auricularia auricula-judae</td><td>AF1 β-1,3-D-glucan main chain with two β-1,6-D-glucosyl residues</td><td>Induces apoptosis of cancer cell</td><td>[30]</td></tr><tr><td>Gymnopus dryophilus (syn. Collybia dryophila)</td><td>β-D-glucan</td><td>Inhibits NO production in activated macrophages</td><td>[31]</td></tr><tr><td>Ophiocordyceps sinensis</td><td>β-D-glucan, heteroglycan, cordyglucan</td><td>Increase in IL-5 induction with decrease in IL-4 and IL-17</td><td>[32]</td></tr><tr><td>Cryptoporus volvatus</td><td>β-1,3-D-Glucan</td><td>Decreases in TLR2 and activate NF-κB</td><td>[33]</td></tr><tr><td>Flammulina velutipes</td><td>Glycoprotein, Flammulina velutipes peptidoglycan (FVP), β-1,3-D-glucan</td><td>Increases NO, IL-1 production, and TNF-α secretion</td><td>[34]</td></tr><tr><td>Ganoderma lucidum</td><td>Ganoderan, Heteroglycan, mannoglucan, glycopeptide</td><td>Stimulates TNF-α, IL-1, IFN-γ production, activate NF-κB.</td><td>[35]</td></tr><tr><td>Grifola frondosa</td><td>Grifolan (1-6-monoglucosyl-branched β-1,3-D-glucan), proteoglycan, heteroglycan, galactomannan</td><td>Macrophage activation, induction of IL-1, IL-6, and TNF-α secretion</td><td>[36]</td></tr><tr><td>Hericium erinaceus</td><td>Heteroglycan, heteroglycan-peptide, β-1,3 branched-β-1,2-mannan</td><td>Induces NO production, increase expression of TNF-α, IL-1β, IL-12</td><td>[15]</td></tr><tr><td>Inonotus obliquus</td><td>β-D-glucan</td><td>Enhance expression of IL-1β, IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) in macrophages</td><td>[37]</td></tr><tr><td>Lentinula edodes (syn. Lentinus edodes)</td><td>Lentinan, glucan, mannoglucan, proteoglycan, β-(1-6)-D-glucan, α-(1-3)-D-glucan</td><td>Induces non-specific cytotoxicity in macrophage and enhance cytokine production Induces cytotoxic effect on a breast cancer cell line</td><td>[38–40]</td></tr><tr><td>Lentinus squarrosulus</td><td>Glucan</td><td>Activation of macrophages, splenocytes and thymocytes</td><td>[41]</td></tr><tr><td>Morchella esculenta</td><td>Galactomannan, β-1,3-D-glucan</td><td>Macrophage activation, activate NF-κB</td><td>[42]</td></tr><tr><td>Morchella conica</td><td>Galactomannan</td><td>Induces NO, IL-1β, IL-6 production</td><td>[43]</td></tr><tr><td>Naematelia aurantialba (syn. Tremella aurantialba)</td><td>Heteroglycan</td><td>Enhances mouse spleen lymphocyte proliferation</td><td>[44]</td></tr><tr><td>Pleurotus sp. 'Florida' (syn. Pleurotus florida)</td><td>α-1,6-glucan and α-1, 3-, β-1,6-D-glucan</td><td>Stimulates macrophages, splenocytes and thymocytes</td><td>[45,46]</td></tr><tr><td>Pleurotus ostreatus</td><td>Pleuran, heterogalactan, proteoglycan</td><td>Induces IL-4 and IFN-γ production</td><td>[47]</td></tr><tr><td>Poria cocos</td><td>β-pachyman, β-Glucan, β-1,3-D-glucan, α-1, 3-D-glucan</td><td>Promotes the immune reaction; increases the expression of cytokines</td><td>[48,49]</td></tr><tr><td>Sarcodon aspratus</td><td>Fucogalactan, 1,6-α-D-glucopyranosyl residue</td><td>Increases the release of TNF-α and NO in macrophage</td><td>[50]</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><td>Source</td><td>Active Compound</td><td>Immunomodulatory Effect</td><td>Refs</td></tr></thead><tbody><tr><td>Schizophyllum commune</td><td>Schizophyllan, 1,6-monoglucosyl branched β-1, 3-D-glucan</td><td>Activation of T cell, increases interleukin, and TNF-α production</td><td>[51]</td></tr><tr><td>Sparassis crispa</td><td>β-Glucan</td><td>Enhances IL-6 and INF-γ production</td><td>[52]</td></tr><tr><td>Taivanofungus camphoratus (syn. Antrodia camphorate)</td><td>β-1,3-D-Gluco-pyranans with β-1,6-D-glucosyl branches, proteoglycan</td><td>Induction of INF-γ, TNF-α</td><td>[53]</td></tr><tr><td>Tropicoporus linteus (syn. Phellinus linteus)</td><td>Acidic polysaccharides</td><td>Activation of murine B cells, Induces IL-12 and IFN-γ production, Blocks NF-κB, TNF-α, IL-1α, IL-1β, and IL-4 production</td><td>[54]</td></tr><tr><td>Trametes versicolor</td><td>Polysaccharide peptide Krestin (PSK), β-1,3-glycosidic bond with β-1,6-glycosidic branches</td><td>Increases the expression of cytokines; stimulates the macrophage phagocytes</td><td>[1,55]</td></tr><tr><td>Tremella fuciformis</td><td>Acidic glucuronoxylomannan α-1,3-D-mannan backbone with β-linked D-glucuronic acid</td><td>Induces human monocytes to express interleukins</td><td>[56,57]</td></tr><tr><td>Macrocybe gigantea (syn. Tricholoma giganteum)</td><td>Polysaccharide-protein complex (PSPC)</td><td>Increases phagocytic function of macrophages by activating macrophages to release mediators such as NO and TNF-α and inhibits S180 and HL-60 cells</td><td>[13,27]</td></tr><tr><td>Xylaria nigripes</td><td>β-Glucan</td><td>Inhibits NO, IL-1β, IL-6, TNF-α, and IFN-γ production</td><td>[58]</td></tr></tbody></table>
3.2. *Mushroom Proteins and Protein-Conjugate Complexes*

Mushroom proteins and protein-conjugate complexes are also well-known as immunomodulatory
compounds. Similar to the polysaccharide-based compounds, these protein-based immunomodulatory
compounds in medicinal mushrooms can also be grouped into different categories. Here, these
compounds are grouped into two major categories: fungal immunomodulatory proteins (FIPs) and
lectins. FIPs differ from lectins by having no conjugate while each lectin contains specific carbohydrates
conjugated to a polypeptide.

Table 3 lists all the lectins from medicinal mushrooms isolated so far that have shown immunomodulatory effects. These lectins have been shown to be capable of stimulating nitrite production, upregulating the expressions of tumor necrosis factor (TNF)-α and interleukins, activating lymphocytes, and promoting the production of macrophage-activating factors etc. The medicinal mushroom species containing such lectins are very diverse, including *Floccularia luteovirens* (syn. *Armillaria luteovirens*), *Ganoderma capense*, *Grifola frondosa*, *Pseudosperma umbrinellum* (syn. *Inocybe umbrinella*), *Pholiota adipose*, *Pleurotus citrinopileatus*, *Russula delica*, *S. commune*, *Leucocalocybe mongolica* (syn. *Tricholoma mongolicum*), *Volvariella volvacea*, and *Xerocomus spadiceus* [59–64]. In addition, several mushroom lectins have also shown potent antiviral, mitogenic, antimicrobial and antioxidant activities [59,63,65–69].

Similarly, a large number FIPs have been identified. The FIP names, the medicinal mushrooms that produce them, and evidence for their specific immunomodulatory effects are presented in Table 4. Among these, the best known is probably Ling-Zhi-8 from *G. lucidum* which acts as an immunosuppressive agent [1]. In addition, aside from immunomodulation, many FIPs have also shown antitumor activities in pharmacological tests, including the inhibition of cell growth and proliferation, the induction of apoptosis and autophagy, and the reduction of invasion and migration of tumor cells. At present, most of these tests are conducted using tissue cultures. Further tests using animal models and clinical trials are needed in order to confirm their safety and efficacy in humans. If confirmed, these FIPs could be more efficiently produced and commercialized through genetic engineering for clinical use.
<table><caption>**Table 3.** Major immunomodulatory lectins from medicinal mushrooms and their immunomodulatory effects.</caption><thead><tr><td>Source</td><td>Lectin name</td><td>Immunomodulatory effect</td><td>Refs</td></tr></thead><tbody><tr><td>Agaricus bisporus</td><td>Agaricus bisporus lectin (ABL)</td><td>Stimulate mice splenocytes mitogenicity and inhibit proliferation of L1210 and HT-29 cells</td><td>[70,71]</td></tr><tr><td>Agrocybe aegerita</td><td>Agrocybe aegerita lectin (AAL)</td><td>Inhibit proliferation of 4T1, HeLa, SW480 SGC7901, MGC803, BGC823, HL-60 and S180 cells</td><td>[72,73]</td></tr><tr><td>Amanita phalloides</td><td>-</td><td>Inhibit proliferation of L1210 cells</td><td>[74]</td></tr><tr><td>Floccularia luteovirens (syn. Armillaria luteovirens)</td><td>Armillaria luteovirens lectin (ALL)</td><td>Stimulate mice splenocytes mitogenicity and inhibit proliferation of L1210, Mouse myeloma MBL2 and HeLa cells</td><td>[75]</td></tr><tr><td>Boletus edulis</td><td>Boletus edulis lectin (BEL)</td><td>Stimulate mice splenocytes mitogenicity and inhibit proliferation of human hepatocyte carcinoma G2 (HepG2) and HT-29 cells</td><td>[76]</td></tr><tr><td>Boletus speciosus</td><td>Boletus speciosus hemagglutinin (BSH)</td><td>Inhibit proliferation of HepG2 and L1210 cells</td><td>[77]</td></tr><tr><td>Clitocybe nebularis</td><td>Clitocybe nebularis lectin (CNL)</td><td>Inhibit proliferation of human leukemic T cells</td><td>[78]</td></tr><tr><td>Flammulina velutipes</td><td>Flammulina velutipes agglutinin (FVA)</td><td>Stimulate mice splenocytes mitogenicity and inhibit proliferation of L1210 cells</td><td>[79]</td></tr><tr><td>Ganoderma capense</td><td>-</td><td>Stimulate mice splenocytes mitogenicity and inhibit proliferation of L1210, M1, HepG2 cells</td><td>[62]</td></tr><tr><td>Grifola frondosa</td><td>Grifola frondosa lectin (GFL)</td><td>Inhibit proliferation of HeLa</td><td>[80]</td></tr><tr><td>Hericium erinaceus (Syn. Hericium erinaceum)</td><td>Hericium erinaceus agglutinin (HEA)</td><td>Inhibit proliferation of HepG2 and human breast cancer MCF7 cells</td><td>[81]</td></tr><tr><td>Kurokawa leucomelas</td><td>Kurokawa leucomelas KL-15</td><td>Inhibit proliferation of U937 cells</td><td>[82]</td></tr><tr><td>Lactarius flavidulus</td><td>Lactarius flavidulus lectin (LFL)</td><td>Inhibit proliferation of HepG2 and L1210 cells</td><td>[83]</td></tr><tr><td>Lignosus rhinocerotis</td><td>Lignosus rhinocerotis lectin (LRL)</td><td>Inhibit proliferation of HeLa, MCF7 and A549 cells</td><td>[84]</td></tr><tr><td>Marasmius oreades</td><td>Marasmius oreades agglutinin (MOA)</td><td>Inhibit proliferation of SW480, HepG2 and NIH-3T3 cells</td><td>[85]</td></tr><tr><td>Pholiota adiposa</td><td>Pholiota adiposa lectin (PAL)</td><td>Inhibit proliferation of HepG2 and MCF7 cells</td><td>[61]</td></tr><tr><td>Pleurotus citrinopileatus</td><td>-</td><td>Stimulate mice splenocytes mitogenicity and inhibit proliferation of S180 cells</td><td>[59]</td></tr><tr><td>Pleurotus eous</td><td>Pleurotus eous lectin (PEL)</td><td>Inhibit proliferation of MCF7, K562 and HepG2</td><td>[86]</td></tr><tr><td>Cerioporus squamosus (syn. Polyporus squamosus)</td><td>Polyporus squamosus lectin 1a (PSL1a)</td><td>Inhibit proliferation of HeLa cells</td><td>[87]</td></tr><tr><td>Pseudosperma umbrinellum (syn. Inocybe umbrinella)</td><td>Inocybe umbrinella lectin (IUL)</td><td>Inhibit proliferation of HepG2 and MCF7 cells</td><td>[60]</td></tr><tr><td>Russula delica</td><td>-</td><td>Inhibit proliferation of HepG2 and MCF7 cells</td><td>[64]</td></tr></tbody></table>
<table><caption>Table 3. *Cont.*</caption><thead><tr><td>Source</td><td>Lectin name</td><td>Immunomodulatory effect</td><td>Refs</td></tr></thead><tbody><tr><td>Russula lepida</td><td>Russula lepida lectin (RLL)</td><td>Inhibit proliferation of HepG2 and MCF7 cells</td><td>[88]</td></tr><tr><td>Schizophyllum commune</td><td>Schizophyllum commune lectin (SCL)</td><td>Stimulate mice splenocytes mitogenicity and inhibit proliferation of KB, HepG2 and S180 cells</td><td>[63,89]</td></tr><tr><td>Stropharia rugosoannulata</td><td>Stropharia rugosoannulata lectin (SRL)</td><td>Inhibit proliferation of HepG2 and L1210 cells</td><td>[90]</td></tr><tr><td>Leucocalocybe mongolica (syn. Tricholoma mongolicum)</td><td>Tricholoma mongolicum lectin 1 (TML-1), Tricholoma mongolicum lectin 2 (TML-2)</td><td>Inhibit proliferation of S180 cells</td><td>[91]</td></tr><tr><td>Volvariella volvacea</td><td>Volvariella volvacea lectin (VVL)</td><td>Inhibit proliferation of S180 cells and enhance IL-2 and IFN-γ transcriptions</td><td>[92,93]</td></tr><tr><td>Xerocomellus chrysenteron (syn. Xerocomus chrysenteron)</td><td>Xerocomus chrysenteron lectin (XCL)</td><td>Inhibit proliferation of NIH-3T3 and HeLa cells</td><td>[94]</td></tr><tr><td>Xylaria hypoxylon</td><td>Xylaria hypoxylon lectin (XHL)</td><td>Inhibit proliferation of HepG2 cells</td><td>[95]</td></tr></tbody></table>
<table><caption>**Table 4.** Major fungal immunomodulatory proteins (FIPs) from medicinal mushrooms and their immunomodulatory effects.</caption><thead><tr><td>FIP Name</td><td>Source</td><td>Immunomodulatory Effect</td><td>Refs</td></tr></thead><tbody><tr><td>FIP-aca</td><td>Taivanofungus camphoratus (Syn. Antrodia camphorate)</td><td>Induce expression of different cytokines (IL-1b, IL-6, IL-12, TNF-α) and chemokines (CCL3, CCL4, CCL5, CCL10)</td><td>[96]</td></tr><tr><td>FIP-cru1</td><td>Chroogomphis rutilus</td><td>Stimulate the proliferation of murine splenocytes and enhanced the secretion of IL-2</td><td>[97]</td></tr><tr><td>FIP-dsq (FIP-dsq2)</td><td>Dichomitus squalens</td><td>Induce apoptosis and interrupt migration of A549 cells</td><td>[98]</td></tr><tr><td>FIP-fve</td><td>Flammulina velutipes</td><td>Stimulate mitogenesis in human peripheral lymphocytes, suppress systemic anaphylaxis reaction, enhance transcription of IL-3, INF-g</td><td>[99]</td></tr><tr><td>FIP-gja</td><td>Ganoderma japonicum</td><td>-</td><td>GenBank: AAX98241</td></tr><tr><td>FIP-gat</td><td>Ganoderma atrum</td><td>-</td><td>[100]</td></tr><tr><td>FIP-glu1 (LZ-8)</td><td>Ganoderma lucidum</td><td>Enhance transcription of IL-2, IL-3, IL-4, IFN-g, TNF-α</td><td>[101]</td></tr><tr><td>FIP-gmi</td><td>Ganoderma microsporum</td><td>Down regulation of TNF-α</td><td>[102]</td></tr><tr><td>FIP-gsi</td><td>Ganoderma sinensis</td><td>Enhance production of IL-2, IL-3, IL-4, INF-g, TNF-a</td><td>[103]</td></tr><tr><td>FIP-gts</td><td>Ganoderma tsugae</td><td>Induce cytokine secretion, cellular proliferation of human peripheral mononuclear cells (HPBMCs), enhance IFN-g expression</td><td>[104]</td></tr><tr><td>FIP-glu2 (LZ-9)</td><td>Ganoderma lucidum</td><td>Activate THP-1 macrophages and induce pro-inflammatory cytokine transcription</td><td>[105]</td></tr><tr><td>FIP-SN15</td><td>Intrageneric shuffled library</td><td>Induce U-251 MG cells apoptosis</td><td>[106]</td></tr><tr><td>FIP-Irh</td><td>Lignosus rhinocerotis</td><td>Inhibit the proliferation of MCF7, HeLa and A549 cancer cell lines</td><td>[84]</td></tr><tr><td>FIP-pcp</td><td>Poria cocos</td><td>Enhance production of IL-1b, IL-6, IL-18, TNF-a, NO</td><td>[107]</td></tr><tr><td>FIP-ppl</td><td>Postia placenta</td><td>Stimulate mouse splenocyte cell proliferation and enhance interleukin-2 (IL-2) release, inhibit proliferation and induce apoptotic effects on gastric tumor cells (MGC823)</td><td>[108]</td></tr><tr><td>FIP-tve2 (FIP-tvc)</td><td>Trametes versicolor</td><td>Increase human peripheral blood lymphocytes, enhanced production of TNF-a, NO</td><td>[109]</td></tr><tr><td>FIP-vvo</td><td>Volvariella volvacea</td><td>Enhance expression of IL-2, IL-4, IFN-g, TNF-a</td><td>[110]</td></tr></tbody></table>

"-" not tested.
### 3.3. Terpenes and Terpenoids

Terpenes are a large and diverse class of hydrocarbon compounds derived biosynthetically from units of isopentenyl pyrophosphate. They are widespread in nature, produced by a variety of plants, particularly conifers, some insects, and fungi, including mushrooms. The addition of functional groups (usually oxygen-containing) to terpenes produce terpenoids. Both terpenes and terpenoids from a number of medicinal mushrooms have shown immunoregulatory activities with medical significance. Table 5 shows the types of terpenes and terpenoids that have been isolated from medicinal mushrooms, including evidence for their specific immunomodulatory activities. For example, *Ganoderma* sp. are known for their high content of triterpenoids and these triterpenoids have shown high immunomodulating and anti-infective activities [111–113]. A study showed that terpenes and terpenoids modulate immune system functions by stimulating the expressions of genes coding for proteins in the nuclear factor (NF)-kB pathway and for mitogen-activated protein kinases [14].
<table><caption>**Table 5.** Major immunomodulatory terpenes and terpenoids from medicinal mushrooms and their immunomodulatory effects.</caption><thead><tr><th>Type of Terpenes</th><th>Source</th><th>Compound</th><th>Immunomodulatory Effect</th><th>Refs</th></tr></thead><tbody><tr><td rowspan="4">Monoterpenoids</td><td><i>Pleurotus cornucopiae</i></td><td>-</td><td>Inhibit the proliferation of HeLa and HepG2 cells</td><td>[114]</td></tr><tr><td><i>Stereum hirsutum</i></td><td>-</td><td>Inhibit the proliferation of HepG2 and A549 cells</td><td>[111]</td></tr><tr><td><i>Inonotus rickii</i></td><td>3α,6α-Hydroxycinnamolide</td><td>Inhibit the proliferation of SW480 cells</td><td>[112]</td></tr><tr><td><i>Pleurotus cornucopiae</i></td><td>Pleurospiroketals A, B, C</td><td>Inhibit the proliferation of HeLa cells</td><td>[113]</td></tr><tr><td rowspan="3">Sesquiterpenoids</td><td><i>Anthracophyllum sp. BCC18695</i></td><td>Anthracophyllone</td><td>Inhibit the proliferation of MCF7, NCI-H187, KB and Vero cells</td><td>[115]</td></tr><tr><td><i>Flammulina velutipes</i></td><td>Enokipodins B, D, J<br>2,5-cuparadiene-1,4-dione<br>Flammulinolides A, B, C, F</td><td>Inhibit the proliferation of HepG2, MCF7, SGC7901, KB, HeLa and A549 cells</td><td>[116,117]</td></tr><tr><td><i>Neonothopanus nambi</i></td><td>Nambinones C</td><td>Inhibit the proliferation of NCI-H187 cells</td><td>[118]</td></tr><tr><td rowspan="4">Diterpenoids</td><td><i>Cyathus africanus</i></td><td>Neosarcodonin O,<br>11-O-acetylcyathatriol, Cyathins H</td><td>Inhibit the proliferation of K562 and HeLa cells</td><td>[119]</td></tr><tr><td><i>Pleurotus eryngii</i></td><td>Eryngiolide A</td><td>Inhibit the proliferation of HeLa and HepG2 cells</td><td>[120]</td></tr><tr><td><i>Sarcodon scabrosus</i></td><td>Sarcodonin G</td><td>Inhibit the proliferation of HOC-21, HEC-1, U251-SP, MM-1CB and HMV-1 cells</td><td>[121]</td></tr><tr><td><i>Tricholoma sp.</i></td><td>Tricholomalide A, B, C</td><td>Inhibit the proliferation of HeLa cells</td><td>[122]</td></tr></tbody></table>
<table><caption>Table 5. *Cont.*</caption><thead><tr><th>Type of Terpenes</th><th>Source</th><th>Compound</th><th>Immunomodulatory Effect</th><th>Refs</th></tr></thead><tbody><tr><td rowspan="10">Triterpenoids</td><td>Ganoderma boninense</td><td>Ganoboninketals A, B, C</td><td>Inhibit the proliferation of A549 and HeLa cells</td><td>[123]</td></tr><tr><td>Ganoderma orbiforme BCC 22324</td><td>Ganoderic acid T and its C-3 epimer compound</td><td>Inhibit the proliferation of NCIH187, MCF7 and KB cells</td><td>[124]</td></tr><tr><td>Ganoderma lucidum</td><td>lucialdehydes B, C, ganodermanondiol, ganodermanonol, ganoderic acid DM, ganoderic acid X</td><td>Inhibit the proliferation of T-47D, LLC, Meth-A, and Sarcoma 180 cells; Decrease the protein levels of CDK2, CDK6, p-Rb, cycle D1 and c-Myc in MCF7 cells; inhibit activity against topoisomerases I and II α and promote apoptosis</td><td>[125–127]</td></tr><tr><td>Ganoderma concinna</td><td>5α-lanosta-7,9(11),24-triene-3β-hydroxy-26-al, 5α-lanosta-7,9(11),24-triene-15α-26-dihydroxy-3-one, 8α,9α-epoxy-4,4,14α-trimethyl-3,7,11,15,20-pentaoxo-5α-pregrane</td><td>Induce apoptosis in promyelocyticleukemia HL-60 cells</td><td>[128]</td></tr><tr><td>Ganoderma tsugae</td><td>Tsugaric acid A, 3β-hydroxy-5α-lanosta-8, 24-dien-21-oic acid</td><td>Inhibit the proliferation of HT-3, T-24, and CaSKi cells</td><td>[129]</td></tr><tr><td>Hypholoma fasciculare (syn. Naematoloma fasciculare)</td><td>Fusciculol C, L, M, G</td><td>Inhibit the proliferation of HCT-15, SK-OV-3, SK-MEL-2 and A549 cells</td><td>[130]</td></tr><tr><td>Astraeus odoratus</td><td>Astraodoric acids A, B, D</td><td>Inhibit the proliferation of KB, NCI-H187, and MCF7 cells</td><td>[131]</td></tr><tr><td>Russula lepida Russula amarissima</td><td>Cucurbitane hydroxyl acid</td><td>Inhibit the proliferation of WISH, CAKI 1 and A549 cells</td><td>[132]</td></tr><tr><td>Leucopaxillus gentianeus</td><td>Cucurbitacin B<br>Leucopaxillone A</td><td>Inhibit the proliferation of MCF7, HepG2, kidney carcinoma CAKI-1 and A549 cells</td><td>[133]</td></tr><tr><td>Hebeloma versipelle</td><td>24(E)-3β-hydroxylanosta-8,24-dien-26-al-21-oic acid</td><td>Inhibit the proliferation of HL60, Bel-7402, SGC-7901 and A549 cells</td><td>[134]</td></tr><tr><td>Tricholoma saponaceum</td><td>Saponaceol A</td><td>Inhibit the proliferation of HL-60 cells</td><td>[135]</td></tr><tr><td>Elfvingia applanata</td><td>The methyl ester of elfvingic acid H</td><td>Inhibit the proliferation of Ehlrich and Kato III cells</td><td>[136]</td></tr></tbody></table>
## 4. Immunomodulation and Other Human Health Effects of Medicinal Mushrooms

The human immune system is tightly linked to tumor development. With the increasing impact of tumor on human health, a large number of studies have been undertaken to identify mushroom extracts/fractions/compounds with antitumor activities. Indeed, some of the observed antitumor activities by medicinal mushroom extracts were based on the activation of the immune system (Tables 2–5). Davis et al. (2020) recently suggested that 17 medicinal mushroom species (*A. brazei*, *Cordyceps militaris*, *Flammulina velutipe*, *F. fomentarius*, *F. officinalis*, *Ganoderma applanatum*, *G. lucidum*, *Ganoderma oregonense*, *G. frondosa*, *Hericium erinaceus* (syn. *Hericium erinaceum*), *I. obliquus*, *L. edodes*, *Tropicoporus linteus* (syn. *Phellinus linteus*), *P. betulinus*, *Pleurotus ostreatus*, *S. commune*) could support both immune-activation for cancer treatments and help resolve host defense-induced inflammatory reactions and facilitate a post-response return to homeostasis for cancer patients. Furthermore, a medicinal mushroom formulation consisting of *G. lucidum*, *L. edodes* and *G. frondosa* showed synergistic antitumor and immuno-modulatory activity in human macrophages [137].

As shown in Tables 2–5, many medicinal mushrooms each can produce different categories of compounds with immunomodulatory effects. Furthermore, different extractions of the same mushroom may show non-overlapping but complementary activities. For example, in *L. edodes*, its heterogalactan (fucomannogalactan) has anti-inflammatory properties [138], lentinan has anti-tumor effect [139], crude water-soluble polysaccharides can activate macrophages and increase the productions of nitric oxide (NO), cytokines, and proteins related to phagocytosis [140], and polysaccharides with both antioxidant effects [141] and antiviral activities [142].

The significance of functional components from medicinal mushrooms has been shown not only from clinical perspectives but also from foods. Because many medicinal mushrooms are commercially cultivated for food, there has been an increasing trend of including mushrooms and their components into other foods to develop functional foods, including adding new flavors or promoting certain types of functions. For example, Ulziijargal et al. used mushroom mycelia of *T. camphoratus*, *A. blazei*, *H. erinaceus*, and *P. linteus* to substitute 5% of wheat flour to make bread. The final product contains substantial amounts of the amino acids gaminobutyric acid (GABA) and ergothioneine and showed beneficial health effects [143]. Kim et al. developed noodles that contained *L. edodes* paste, resulting in a higher quality, fibre-rich functional food with antioxidant and hypocholesterolemic properties [144]. Components of other mushrooms, e.g., *Pleurotus sajor-caju* dry powder, *A. bisporus* extracts, the freeze-dried powder from *A. aegerita*, *Suillus luteus*, and *Coprinopsis atramentaria* have been used to develop snacks and cheese-related products with much commercial success [145–148]. The benefits include increased proteins, minerals, crude fiber or ingredients with antioxidant potentials and free radical scavenging capacities. The diversified flavors and tastes as well as the enhanced nutritional values of food due to the addition of mushroom components represent an exciting direction for the edible and medicinal mushroom industries.

## 5. Mechanisms for the Immunomodulation Effects of Medicinal Mushroom Compounds

The immune system consists of a network of cells, tissues and organs that work together to defend the body against attacks by “foreign” invaders [2]. The network is connected by lymphatic vessels from organ to organ. The network includes protective barriers that constantly communicate with lymphatic fluid rich in white blood cells and leukocytes. When pathogens break our physical barriers (i.e., skin and mucosal membranes of the mouth, nose, the gastrointestinal tract, and the urogenital tract), the next line of the body’s defense response is activated. This line of defense includes granulocytes and monocytes that also function as antigen-presenting cells (APCs) for helper T lymphocytes. These cells synthetize and secrete lipid mediators such as prostaglandins as well as cytokines which act as messengers in regulating immune response and stimulating adaptive immunity. For example, natural killer (NK) cells can recognize infected and abnormal cells, such as cancer cells and kill these cells by inducing them to undergo apoptosis or by producing cytokines, such as interferon-gamma (IFN-γ). They also
activate macrophages and kill phagocytosed microbes. Figure 3 presents the overview of the key immune response against microbial pathogens.

<figure><img src="image_147.png" /><figcaption>**Figure 3.** Key components and pathways in host immune response against pathogen infections.</figcaption></figure>

It is well known that the human immune system can be modulated by foods, supplements or endogenous bioactive agents [2]. Different types of immunoregulatory compounds have been isolated from medicinal mushrooms, including mushroom fruiting bodies and fermented mycelia. Tables 2–5 present the specific components isolated from different mushroom species that have shown significant immunomodulatory activities, including their (potential) mechanisms of action. For example, two polysaccharides from two different mushroom species have shown significant immunoenhancing activities. In the first, a glucuronoxylomannan TAP-3 obtained from *Naematelia aurantialba* (syn. *Tremella aurantialba*) showed marked immune enhancement activity and promoted NO, IL-1β and TNF-α secretions from macrophages [149]. Similarly, another study showed that at a concentration of 40 µg/mL, polysaccharide CCP from *Craterellus cornucopioides* strengthened the phagocytic function of macrophages, increased the expression of cytokines, upregulated the expression of cell membrane receptor TLR4 and downstream protein kinase products through activation of the TLR4-NFκB pathway [150].

Some of these bioactive compounds can also directly attack cancer cells while showing immunoregulatory effects. For example, Li et al. reported that polysaccharide LRP-1 purified from *Leccinum rugosiceps* inhibited the growth of human hepatoma cells HepG2 and human breast carcinoma MCF-7 cells, and induced the secretion of NO, IL-6 and TNF-α in vitro [151]. Similarly, a recent report showed that an aqueous extract of *Sarcodon imbricatus* (SIE) effectively inhibited the growth, migration, and invasion properties of breast cancer cells in vitro and reduced tumor growth in vivo, while showing increased expression of PD-L1 and increased NK cell viability [152]. Furthermore, Xue et al. reported that a triterpenoid EAe from *Pleurotus eryngii* inhibited MCF-7 cell lines proliferation with an EC50 of 298.26 µg/mL, and significantly inhibited the growth of CD-1 tumors (inhibition rate of 65%) in mice in a dose-dependent manner without toxicity [153].

## 6. Relationship between Structure and Activity of Immunomodulatory Compounds from Medicinal Mushrooms

Immunomodulators from medicinal mushrooms have been shown to be capable of stimulating both innate and adaptive immune responses. They activate innate immune system components such as natural killer (NK) cells, neutrophils, and macrophages, and stimulate the expression and secretion of cytokines. These cytokines in turn activate adaptive immunity by promoting B cell proliferation and
differentiation for antibody production and by stimulating T cell differentiation to T helper (Th) 1 and Th2 cells, which mediate cellular and humoral immunities, respectively [154].

As shown in Tables 2–5, a large number of immunoregulatory compounds from medicinal mushrooms have been reported. These compounds differ greatly in their molecular weight and structure. Below we describe the relationships between their molecular structures and immunoregulatory activities.

## 6.1. Polysaccharides

Polysaccharides are the most commonly reported natural immunomodulators from mushrooms. The immunomodulating polysaccharides are highly diversified in their sugar compositions, main chain polymer structures, degrees of branching, conformations, molecular weights, and other physical properties, which together have significant effects on the bioactivity and mode of action of each polysaccharide [18]. These polysaccharides are either homoglycans (polysaccharides that contain residues of only one type of monosaccharide molecule) or heteroglycans (polysaccharides that contain residues of two or more types of monosaccharide molecules), and are able to combine with other molecules such as oligo- or poly-peptides to make peptidoglycan or polysaccharide-protein complexes. In general, higher molecular weight polysaccharides exhibit greater bioactivity [155]. These large polysaccharides are not able to penetrate the immune cells, but instead act to bind cell receptors. For example, the highest immunomodulatory activity of PSK was associated with the highest molecular weight fraction of this compound, at >200 kDa [156]. Similarly, the highest activity of a polysaccharide fraction of *G. frondosa* extract was ascribed to one with a molecule weight of over 800 kDa [157]. In contrast, low molecular weight polysaccharides can penetrate immune cells and exert stimulatory effects from within.

The number and lengths of short branched chains in mushroom polysaccharides can significantly influence their bioactivity [155]. In most cases, the bioactive immunomodulator polysaccharides are characterized by a main chain of 1,3-β-D-glucan with a small number of short branched chains with 1,6-β-linkage (Figure 4). Studies have shown that immunologically active polysaccharides generally have a degree of branching number (DB) between 20% and 40%. For example, lentinan has a DB number of 40%, schizophyllan of 33%, and PSK of 20%. While a high DB number is generally correlated with a high activity, in some cases, debranching of polysaccharides can also increase their bioactivity. For example, the partially debranched form of pachymaran from *Poria cocos* showed greater activity than the original native form [158]. Even in the well-studied lentinan, its maximal immunomodulating and antitumor activities were achieved when the molecule had a DB of 32% [159], and there was a negative correlation between their biological activity and DB number between 32 and 40% [160].

<figure><img src="image_148.png" /><figcaption>**Figure 4.** The basic structures of bioactive polysaccharides 1,3-β-D-glucan and short branched chains with 1,6-β-linkage.</figcaption></figure>

Aside from the main-chain structure and branching pattern, the conformation of polysaccharides can also impact their bioactivity, e.g., by influencing the stability of the structure. In polysaccharides, the triple helix conformation is usually more stable than other conformations and bears the cytokine stimulating activity of the β-D-glucan. Lentinan, schizophyllan, scleroglucan, and PSK all have a triple helix structure [161]. However, not all polysaccharide immunomodulators from mushrooms have the
triple helix structure. For example, mushroom polysaccharides with a random coil conformation can also have potent immunomodulating and anticancer activity [56].

Chemical modification is an effective and common approach to increase biological activities of polysaccharides. This approach has been applied to develop a number of effective immunomodulators from mushroom polysaccharides. Those modifications include carboxymethylation, hydroxylation, formyl-methylation, amino-ethylation, or sulfation. The introduction of such chemical groups may increase the possible contacts between the modified polysaccharides and the immune cell receptors through hydrogen bonding and/or electrostatic attraction, and thus increase the immunological response. For example, the sulfated cell wall glucan from *L. edodes* exhibited higher immunomodulatory and anticancer activities compared to the native polysaccharides [162]. The increased effect from sulfation may be related to the increased solubility, as shown in the hyper branched β-glucan TM3b. Taken together, molecular weight, branching, chemical configuration, and chemical modification can all have strong influence on the bioactivity of polysaccharides from mushrooms.

## 6.2. Lectins

Lectins belong to a unique group of proteins that can recognize and interact with various cell surface carbohydrates/glycoproteins. Mushroom lectins have shown specific immunomodulatory, antiproliferative, and antitumor activities [163]. The diverse sources of mushroom lectins have different immunomodulatory mechanisms: some mediate their actions by activating the immune system while others produce potent cytotoxic effects towards cells [91]. For example, two lectins extracted from *L. mongolica* (syn. *T. mongolicum*), TML-1 and TML-2, show immunomodulatory and antitumor activities. These two lectins stimulate the production of nitrite and tumor necrosis factor (TNF)-α and inhibit the growth of mouse lymphoblast-like (p815) mastocytoma cells by the production of macrophage-activating factors. These factors include interferon (IFN)-γ and other cytokines, activated through upregulation of inducible nitric oxide synthase (NOS), interleukin (IL)-1β, and transforming growth factor-β [91]. *G. frondosa* lectin is reported as having a potent cytotoxic effect against HeLa cells in vitro, even at very low concentrations. A 15.9-kDa homodimeric, lactose-binding, ricin-B-like lectin (CNL) from *Clitocybe nebularis* exhibited antiproliferative activity against human leukemic T cells [78], which induces the maturation and activation of dendritic cells (DCs) and stimulates several proinflammatory cytokines such as IL-6, IL-8, and TNF-α [164]. The encoding gene of CNL from *C. nebularis* has been cloned and successfully expressed in *Escherichia coli* [165].

## 6.3. FIPs

The fungal immunomodulatory proteins are a group of proteins with highly similar amino acid sequences. They exist as dimers in a dumbbell-shaped structure similar to that of the variable region of immunoglobulin heavy chains [166]. The FIPs have shown diverse functions. Through binding to Toll-like receptors (TLRs), FIPs stimulate antigen presenting cells and release cytokines such as NO and IL-12. By activating phosphorylation of p38/MAPK and increasing the production of NF-κB, FIPs can promote the proliferation and differentiation of helper T cells (Th0) to form Th1 cells and Th2 cells, activate macrophages and B cells, produce a variety of cell factors (Figure 5). For example, FIP-fve from *Flammulina velutipes* can upregulate the expression of intercellular adhesion molecules on the T cell surface by phosphorylation of p38/MAPK, and activate Th1 cells to produce IL-2, IFN-γ, to exert its immunomodulatory effect [99]. FIP-vvo can not only activate Th1 cells and enhance IL-2, TNF-α and IFN-γ transformations, but also induce Th2 cells to produce IL-4, B cell differentiation, and the transformation of immunoglobulin and production of antibody IgE. Several studies have also shown that by interacting with TLRs, FIP can activate other signaling pathways besides the p38/MAPK and NF-κB. For example, FIP from *Ganoderma tsugae* (FIP-gts) can stimulate human peripheral blood monocyte to produce IFN-γ and activates the PI3K/Akt signaling pathway [104].
<figure><img src="image_149.png" /><figcaption>**Figure 5.** FIPs immunomodulatory mechanism by toll-like receptors (TLRs) signaling pathway.</figcaption></figure>

FIPs typically exist in low quantities in their native mushrooms. The low yield/production has been a major limitation of their research and application. Therefore, techniques are being rapidly developed to enhance the production of recombinant FIPs in other organisms such as the yeast *Pichia pastoris* and the bacterium *E. coli*. For example, the LZ-8 gene of *G. lucidum* has been expressed in *P. pastoris* to produce a recombinant LZ-8 protein (rLZ-8). While the recombinant protein lacks the carbohydrate moiety of the native protein, it shows similar bioactivity for IL-2 induction as the native protein. The FIP-fve protein has also been successfully expressed in *E. coli* [1]. Interestingly, the recombinant FIPs showed higher immunomodulatory activity and induced greater expression of specific cytokines than that extracted from the mushrooms [167].

## 6.4. Terpenes and Terpenoids

Terpenes and terpenoids are widely distributed in mushrooms. They are a large and diversified group of organic compounds but share the core of isoprene five-carbon atom units of molecular formula (C₅H₆)n as the main building block (Figure 6) [1,13]. Among this group of compounds, the best-known is probably the triterpenoids from *G. lucidum* and *G. lingzhi*. These triterpenoids can help reduce drug nephrotoxicity and minimize inflammation. Figure 6 shows a diversity of terpene derivatives in *G. lucidum* and *G. lingzhi*, including ganodermic and ganoderic acids, ganoderals, ganoderols, ganodermanontriol, lucidone, and ganodermanondiol. All these compounds have shown immunomodulating, antitumor, and/or anti-infective activities [168]. At present, their mechanisms of action and structure-activity relationships are little understood. However, their broad activities suggest significant potential for research and for clinical therapeutic applications.
<figure><img src="image_150.png" /><figcaption>**Figure 6.** The structures of representative terpenes and terpenoids in the fungus *Ganoderma lucidum* and *Ganoderma lingzhi*.</figcaption></figure>

## 7. Genomes and Molecular Techniques in the Study of Immunomodulatory Compounds in Medicinal Mushrooms

Due to their environmental, agricultural, commercial, and/or medical interests, the genomes of a number of medicinal mushrooms have been sequenced and annotated. Table 6 lists the genomic features of 12 representative medicinal mushrooms. These species differ in genome size and/or gene content. Analyses of these genomes have revealed some of the genes related to the synthesis and production of immunomodulatory compounds in medicinal mushrooms. Not surprisingly, the most commonly identified genes related to immunomodulatory effects are those coding for FIPs (Table 6). However, in *G. lucidum*, genes involved in the synthesis of several other immunomodulators have also been identified. The identification and confirmation of those genes require genetic manipulation systems which are not available at present for most medicinal mushrooms. In *G. lucidum*, such a system is available.

Ganoderic acids (GAs) are among the main active ingredients of *G. lucidum* with immunomodulatory effects. GAs belong to the triterpenoid secondary metabolites. Genome sequence analyses and functional studies showed that the terpenoids in *G. lucidum* are synthesized through the Mevalonate (MVA) pathway. Several genes in this pathway in *G. lucidum* have been cloned and their functional roles confirmed, including those encoding 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) and Farnesyl diphosphate synthase (FPPs). Genome sequence data mining also identified the putative genes involved in the modification of the triterpene backbone, such as those involved in cyclization and glycosylation, which are very important for the synthesis of the diversity of GAs in *G. lucidum*.

At present, most of the genes and metabolic pathways involved in the synthesis of immunomodulators in the categories of polysaccharides, lectins, and terpenoids in medicinal mushrooms have not been identified or confirmed. However, the availability of increasing genomic resources coupled with the broad pharmacological activities and therapeutic effects of medicinal mushrooms should help facilitate the identification of genes and metabolic pathways involved in their biosynthesis. Such understandings could help future productions of those compounds through biotechnology using surrogate hosts.
<table><caption>**Table 6.** Major genomic features of representative medicinal mushrooms.</caption><thead><tr><th>Medicinal Mushroom</th><th>Genome Size Mb</th><th>Number of Genes</th><th>GC Content (%)</th><th>Known Genes Related to Immunomodulatory Effects</th><th>Genetic Manipulations (Transformation Method)</th><th>Refs</th></tr></thead><tbody><tr><td>Agrocybe aegerita</td><td>44.7908</td><td>14110</td><td>49.2</td><td></td><td>Polyethylene glycol-mediated transformation (PEG)</td><td>[169]</td></tr><tr><td>Agaricus bisporus</td><td>30.78</td><td>10863</td><td>46.5</td><td></td><td>PEG, Electroporation, Particle bombardment, Agrobacterium tumefaciens-mediated transformation (ATMT)</td><td>[170–173]</td></tr><tr><td>Flammulina velutipes</td><td>35.64</td><td></td><td>49.76</td><td>Fip-fve</td><td>PEG, Electroporation, Electro-injection, Restriction enzyme-mediated integration (REMI), ATMT</td><td>[173–177]</td></tr><tr><td>Ganoderma atrum</td><td></td><td></td><td></td><td>fip-gat</td><td></td><td>[178]</td></tr><tr><td>Ganoderma lucidum</td><td>43.68</td><td></td><td>55.4</td><td>fip-glu, Mevalonate (MVA) pathway genes: AACT (acetyl-CoA acetyltransferase); HMGS (3-hydroxy-3-methylglutaryl-CoA synthase), HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), MPK (phosphomevalonate kinase), MVD (pyrophosphomevalonate decarboxylase), IDI (isopentenyl-diphosphate isomerase), GPPs (geranyl diphosphate synthase), FPPs (farnesyl diphosphate synthase), SQS (squalene synthase), SE (squalene monooxygenase), OSC (2,3-oxidosqualene-lanosterol cyclase), P450 (cytochrome P450), UGTs (uridine diphosphate glycosyltransferases)</td><td>PEG, Electroporation, REMI</td><td>[173,179–181]</td></tr><tr><td>Ganoderma sinensis</td><td>48.96</td><td>15478</td><td>55.6</td><td>fip-gsi</td><td></td><td>[103,182]</td></tr><tr><td>Ganoderma tsugae</td><td>45.5</td><td></td><td></td><td>fip-gts</td><td></td><td>[183]</td></tr><tr><td>Hericium erinaceus (syn. Hericium erinaceum)</td><td>41.21</td><td></td><td>52.43</td><td></td><td>ATMT</td><td>[184,185]</td></tr><tr><td>Lentinula edodes</td><td>39.92</td><td>12051</td><td>46</td><td></td><td>PEG, Electro-injection, REMI, ATMT</td><td>[173,186]</td></tr><tr><td>Pleurotus ostreatus</td><td>34.36</td><td>12296</td><td>50.76</td><td></td><td>PEG, Electroporation, REMI, Particle bombardment</td><td>[173,187–189]</td></tr><tr><td>Postia placenta</td><td>66.6724</td><td>12716</td><td>47.2</td><td>fip-ppl</td><td></td><td>[108,190]</td></tr><tr><td>Trametes versicolor</td><td>44.794</td><td>14572</td><td>57.3</td><td>fip-tvc</td><td></td><td>[191,192]</td></tr><tr><td>Volvariella volvacea</td><td>35.72</td><td></td><td>48.8</td><td>fip-vvo</td><td>PEG, Particle bombardment, ATMT</td><td>[193,194]</td></tr></tbody></table>
8. Conclusions and Perspectives

Many edible and medicinal mushrooms contain compounds with significant immunoregulatory activities. This paper attempts to provide a comprehensive review on the types of these compounds; their distributions, structures and functions; and their potential mechanisms of actions. These compounds have shown their activities through in vitro, ex vivo, tissue cultures, and/or in vivo studies. Some of these compounds have been commercialized and licensed for clinical use.

Aside from the above described compounds, other compounds from edible and medicinal mushrooms may also have great potentials. One such group is chitin and chitin-related compounds. Chitin is the most common aminopolysaccharide polymer in nature and the main material that gives strength to the fungal cell walls (as well as to the exoskeletons of crustaceans and insects). Through deacetylation, either chemically or enzymatically, chitin can be converted to chitosan, a well-known derivative. Through hydrolysis, both chitin and chitosan can be converted to chito-oligosaccharides. Several fungal chitin, chitosan, and chito-oligosaccharides have shown promising benefits to humans and human health [195,196]. For example, chitins from filamentous molds such as *Aspergillus niger* and *Mucor rouxii* and other organisms have been used in plant protection and food processing; chitosan in diagnosis, drug delivery, infection control, molecular imaging, and wound healing; and chito-oligosaccharides in antimicrobial and antitumor activities [195,196]. At present, none of those tested chitin, chitosan, and chito-oligosaccharides for human effects have come from edible or medicinal mushrooms yet. However, due to the similar chemical structures of chitin from different groups of organisms, it's highly likely that this group of natural products from edible and medicinal mushrooms will have similar effects and they represent a promising area of future development for edible and medicinal mushrooms.

While the future looks bright, significant issues remain before the full potential of medicinal mushrooms can be reached. Specifically, during our review of the literature, we identified several significant gaps and areas for future research and development. In the first, there is an urgent need to identify the structures and mechanisms of action for active ingredients in many extracts and formulations from medicinal mushrooms. More rigorous chemical analyses as well as understanding the in vivo pharmacokinetics and pharmacodynamics of individual compounds are needed to fill this gap of knowledge. The second promising area of study is to identify the genes and metabolic pathways involved in producing these immunomodulators in medicinal mushrooms. As shown above, aside from the few FIPs where the specific encoding genes have been identified, we have little information about the genes and how they are regulated in producing the other types of mushroom immunomodulators. While the availability of high through-put technologies and genome sequences are facilitating the discoveries, experimental investigations are needed in order to confirm and identify the conditions for increased productions of these compounds. Fortunately, gene editing technologies and -omics tools are becoming increasingly accessible to the broader life sciences communities. In the third, most immunomodulators described above exist in low quantities in medicinal mushrooms and their extractions can take a long time and be costly. For efficient production, it is very important to develop alternative approaches, e.g., by cloning and expressing the relevant biosynthesis genes in alternative hosts, using industrial fermentations, and developing efficient extraction and purification protocols from such commercial cell cultures. Lastly, the potential interactions between immunomodulators from mushrooms and other medicines, foods, and food supplements need to be critically analyzed in order to establish guidelines for safe and effective use of immunomodulators from medicinal mushrooms [197]. Indeed, at present, safety data about many medicinal mushroom products are not available from controlled clinical trials and associated negative side effects have been reported in several cases for certain types of usages of medicinal mushroom products [198,199]. There is also a cultural difference between Oriental and Western cultures about the use of medicinal mushrooms [197], presenting both a challenge and an opportunity for researchers and policy makers on the broad implications of these products on human health.
**Author Contributions:** S.Z. and J.X. conceived and prepared the manuscript, Q.G., C.R. and Z.Z. participated in the manuscript editing; S.W. and Y.L. provided the photos of mushrooms. All authors have read and agreed to the published vision of manuscript.

**Funding:** This work was supported by National Natural Science Foundation of China (NSFC31701975), by the Collaborative Innovation Center of Beijing Academy of Agricultural and Forestry Sciences (KJCX201915), and by Beijing Academy of Agriculture and Forestry Sciences (KJCX20200208).

**Acknowledgments:** We thank Junliang Zhou from Kunming University for providing the photos of mushroom species.

**Conflicts of Interest:** The authors declare no conflict of interest.

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Review

# Sphinganine-Analog Mycotoxins (SAMs): Chemical Structures, Bioactivities, and Genetic Controls

Jia Chen<fnref n="1" />, Zhimin Li<fnref n="1" />, Yi Cheng<fnref n="1" /> ID, Chunsheng Gao<fnref n="1" />, Litao Guo<fnref n="1" />, Tuhong Wang<fnref n="1" /> ID
and Jianping Xu<fnref n="1" />,<fnref n="2" />,* ID

Received: 31 October 2020; Accepted: 22 November 2020; Published: 24 November 2020

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updates

**Abstract:** Sphinganine-analog mycotoxins (SAMs) including fumonisins and *A. alternata* f. sp. *Lycopersici* (AAL) toxins are a group of related mycotoxins produced by plant pathogenic fungi in the *Fusarium* genus and in *Alternaria alternata* f. sp. Lycopersici, respectively. SAMs have shown diverse cytotoxicity and phytotoxicity, causing adverse impacts on plants, animals, and humans, and are a destructive force to crop production worldwide. This review summarizes the structural diversity of SAMs and encapsulates the relationships between their structures and biological activities. The toxicity of SAMs on plants and animals is mainly attributed to their inhibitory activity against the ceramide biosynthesis enzyme, influencing the sphingolipid metabolism and causing programmed cell death. We also reviewed the detoxification methods against SAMs and how plants develop resistance to SAMs. Genetic and evolutionary analyses revealed that the *FUM* (fumonisins biosynthetic) gene cluster was responsible for fumonisin biosynthesis in *Fusarium* spp. Sequence comparisons among species within the genus *Fusarium* suggested that mutations and multiple horizontal gene transfers involving the *FUM* gene cluster were responsible for the interspecific difference in fumonisin synthesis. We finish by describing methods for monitoring and quantifying SAMs in food and agricultural products.

**Keywords:** sphinganine-analog mycotoxins; fumonisins; AAL-toxin; chemical structure; toxicity; genetics and evolution; biosynthesis

## 1. Introduction

Mycotoxins are secondary metabolites produced by various fungi. These metabolites have important ecological functions on living systems in their natural habitats. As secondary metabolites, mycotoxins are regarded as not essential for fungal growth or reproduction. However, their toxic effects to plants, animals, as well as humans are attracting increasing attention from chemists, biologists, food scientists, and healthcare professionals. Many fungi are capable of synthesizing mycotoxins, including certain saprophytic molds, poisonous mushrooms, human fungal pathogens, and plant fungal pathogens. Mycotoxins produced by plant pathogenic fungi can be divided into two groups: (i) host-selective (or host-specific) toxins (HSTs) and (ii) non-host-specific toxins (nHSTs), depending on whether they are specifically toxic to host plant (HSTs) or to a wide range of species (nHSTs). The known mycotoxins are typically low molecular-weight chemicals but with diverse structures and modes of actions. One group of mycotoxins are structurally analogous to sphingosine, the backbone precursor of sphingolipids that play essential structural and cellular roles in eukaryotic cells. These toxins are called

<footnote n="1">¹ Institute of Bast Fiber Crops and Center of Southern Economic Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China; chenjia01@caas.cn (J.C.); lizhimin@caas.cn (Z.L.); chengyi@caas.cn (Y.C.); gaochunsheng@caas.cn (C.G.); guolitao@caas.cn (L.G.); wangtuhong@caas.cn (T.W.)</footnote>

<footnote n="2">² Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada</footnote>

<footnote>* Correspondence: jpxu@mcmaster.ca</footnote>
sphinganine-analog mycotoxins (SAMs), with fumonisins and the *Alternaria alternata* f. sp. *Lycopersici* (AAL) toxins as the two most widely studied groups of SAMs. SAMs are toxic to plants and animals. They act by inhibiting the ceramide synthase (CerS), thereby influencing the sphingolipid metabolism and initiating apoptosis in animals and programmed cell death (PCD) in plants [1–3]. The objective of this paper is to provide an updated review on the structural diversity, syntheses, modes of action, and health impacts of SAMs.

The discovery of fumonisin was first reported in 1988 and the organism producing it was *Fusarium verticillioides* (syn. *Gibberella fujikuroi* mating population A, syn. *G. moniliformis* Wineland, syn. *F. moniliforme* Sheldon) [4]. Fumonisins have since been found to be produced by at least 18 species of the *Fusarium* genus, with *F. verticillioides* and *F. proliferatum* being the most prominent, and by three unrelated fungal genera, *Aspergillus* section *Nigri* (such as *Asp. niger*, *Asp. Welwitschiae* (syn. *Asp. awamori*) and so on, known as black aspergilli), *Tolypocladium* (*T. inflatum*, *T. cylindrosporum*, and *T. geodes*), and *Alternaria* (the tomato pathotype of *A. alternata*, formerly known as *A. arborescens*) [5–10]. Species of the *Fusarium* genus can be found as saprophytes in soil and as endophytes and pathogens of many plants worldwide. A common group of diseases caused by *Fusarium* pathogens is rotting that can happen to all tissues during all stages of plant development [11,12]. In addition, the *Fusarium* species can infect crops at the post-harvest period during storage [13]. The fungal propagules surviving in the soil can also infect new crop plants and can be carried to new fields by wind or by anthropogenic activities, such as when seedlings are transplanted [14]. *Fusarium* strains can synthesize fumonisins during all stages of their growth, including the saprophytic stage in the soil, during their pathogenesis, and as endophytes in different parts of plants, as well as during crop storage after harvest [15].

Fumonisins, as a nHST, are major contaminants of cereals and grains, including corn, rice, wheat, barley, rye, oat, millet, and products made based on these crops [16]. The consumption of food contaminated by fumonisins significantly increases health problems for humans, leading to a variety of cancers such as esophageal cancer and neurological defects [17,18]. For example, the International Agency for Research on Cancer (IARC) characterized fumonisin FB₁ as a group 2B carcinogens for humans [16]. Fumonisins can also cause diseases and adverse effects in other species, especially in livestock when the feeds are contaminated [19]. Well-known diseases in livestock caused by fumonisins include leukoencephalomalacia in horses and pulmonary edema syndrome in pigs [20,21].

Similar to fuminisins, the AAL-toxins include a family of structurally analogous metabolites. AAL-toxins are a group of HST produced by the ascomycete fungal pathogen *A. alternata* f. sp. *Lycopersici*, the causal agent of tomato stem canker disease [22]. It should be noted that several other pathotypes of *A. alternata* could also produce other HSTs responsible for fungal pathogenesis on their specific host plants, respectively [23]. Unlike other HSTs produced by *A. alternata*, besides the susceptible tomato host, AAL-toxins can also affect many other weeds and crops of dicotyledonous species and at least 25 species of *Solanaceae* [24,25]. Furthermore, the tomato pathotype of *A. alternata* was also reported to produce fumonisins B (FBs) [8,26]. AAL-toxin and FBs were not only detected in the necrosis plant tissues and culture media inoculated by *A. alternata* but also in spores and mycelia of this pathogen [27]. However, AAL-toxin remains the only toxin as a pathogenicity factor for stem canker disease of sensitive tomato varieties, while fumonisins are toxigenic virulence factors [28].

Because of the adverse impacts of SAMs on animal and human health, these toxins are also attracting increasing attention from food inspectors and public health agencies. Over the last three decades, significant progress has been made in our understanding of SAMs. Our objectives of this review are to capture these developments on SAMs with regard to their chemical structural diversity, the relationship between structure and activity, PCD induction, detoxification, genetics and evolution of SAMs biosynthesis, and laboratory detections.
## 2. Chemical and Structural Properties

### 2.1. Chemical and Structural Properties of Sphingolipids

SAMs have a distinct structural similarity to sphinganine (Figure 1). Sphinganine (dihydrospingosine, DHS) is the simplest class of sphingolipids and has a backbone that consists of a linear aliphatic group with 18-carbon, an amino at C-2, and two hydroxyls (-OH) at C-1 and C-3, respectively. Phytosphingosine is obtained if a hydroxyl is introduced at C-4. Sphingosine consists of the sphinganine backbone but with a double bond at C-4. Ceramides are synthesized by linking an amide fatty acid at C-2 of sphingosine. Ceramides is a waxy lipid molecule, which is found in high concentrations in the membrane of eukaryotic cells. More complex sphingolipids can be formed by linking different chemical groups to hydroxyl (C1) of ceramides. Sphingolipids are one type of lipids widely found in their membranes in eukaryotes and a few prokaryotes, and they form complex and diverse interactions with other molecules [29]. Sphingolipids play important structural and functional roles, they are involved in a variety of signal transductions and crucial cellular processes [30,31]. For example, in humans, ceramides help form the skin’s barrier and regulate immune response, protecting the skin against environmental irritants, pollutants, and water loss. Without the proper ratio of ceramides on our epidermal cells, the barrier of the skin will be damaged, resulting in dryness, itching, and irritation [32].

<figure><img src="image_155.png" /><figcaption>**Figure 1.** Chemical structure of sphingolipids. The table shows the different substituents in the chemical scaffold of the most essential sphingolipid.</figcaption></figure>

### 2.2. Chemical and Structural Properties of Fumonisins

SAMs consist of two main types of toxins, fuminisins and AAL-toxins. Fumonisins can be divided into seven groups (FA, FB, FC, FD, FP, FP<sub>y</sub>, and FL<sub>a</sub>). These groups differ in the nitrogen functional group and the length of the carbon backbone [5]. Most fumonisins contain a 19–20 (FD contain 17 or 18 carbon) linear backbone similar to sphinganine with one nitrogen functional group (except for FP<sub>y</sub>s and FL<sub>a</sub>s), two to four hydroxyl, two methyl, and two propane-1,2,3-tricarboxylic acid (PTCA) side chains esterified to the backbones [26,33]. The structural features of the seven groups of fumonisins are shown in Figure 2. Among them, the B group is the dominant one. For example, FB<sub>1</sub> accounts for 70–80% of the total fumonisins produced by *F. verticillioides* and is the predominant toxic form [5]. FB<sub>2</sub> and FB<sub>3</sub> are isomers of each other but with one less hydroxyl group than FB<sub>1</sub>. The B series of fumonisins (FBs) are also the main food contaminants. Group A fumonisins (FA) are acetylated derivates of group B toxins, with lower toxicity and bioactivity than their FB counterparts [34]. Group C fumonisins (FC) have the same nitrogen functional group as FB<sub>1</sub> but lack the terminal methyl group at C-1 [35]. Three forms of acetylated FC<sub>1</sub> have been discovered in *F. oxysporum* [36]. Group P fumonisins (FP) have a nitrogen functional group of 3-hydroxypyridinium instead of the amino group in FB at the R<sub>2</sub> position [37]. The FC and FP groups have similar phytotoxic and cytotoxic effects to those caused by FB<sub>1</sub> or AAL-toxin [38]. Aside from these four main groups, there are several other lesser-known fumonisin analogs, with one or two PTCA replaced by a hydroxyl or carbonyl or other carboxylic acids group at C-13 and/or C-14 of the backbone (for example, HFB<sub>1</sub>, as show in
Figure 2). Rheeder et al. summarized the 28 fumonisin analogs that have been characterized between 1988 and 2002 [5]. By reversed-phase high-performance liquid chromatography/electrospray ionization ion trap multistage mass spectrometry (RP-HPLC/ESI-IT-MSn), Bartok et al. detected 58 fumonisins (including FD) or fumonisin-like compounds from *F. verticillioides* in rice cultures, and 28 isomers of FB₁ [33,39]. Indeed, the recent application of a semi-targeted method revealed over 100 structurally related compounds from SAMs-producing fungi, including a hydroxyl-FB₁, and two new classes of non-aminated fumonisins (FPys and FLas) [26].

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<figure><img src="image_157.png" /><figcaption>**Figure 2.** Chemical structure of sphinganine-analog mycotoxins (SAMs). Panel A shows the AAL-toxins, Panel B shows fumonisin. In the table of each panel, the different substituents present in the chemical scaffolds of individual compounds are shown.</figcaption></figure>

## 2.3. Chemical and Structural Properties of AAL-Toxin

The AAL-toxins have a structural similarity to fumonisins (Figure 2). The main difference between fumonisins and AAL-toxins is that AAL-toxins have one fewer PTCA side chain than fumonisins. The AAL-toxins have been divided into five pairs based on their side chain structures: A, B, C, D, and E pairs (TA, TB, TC, TD and TE). These pairs differ in their nitrogen functional group and hydroxylation at C-4 or C-5 positions of the backbone [40–42]. Each pair of AAL-toxins is composed of two regioisomers
with PTCA esterified to C-13 or C-14 of the backbone, respectively. The TA pair is the major pair of toxins, with the TB and TC pairs formed by removing hydroxyl groups one by one from C-5 and C-4 of the TA pair. The TD and TE pairs were acetylated derivatives of TB and TC respectively, while the acetylated form of TA and keto derivatives of AAL-toxins (2-keto or 14-keto analogues predicted) were also found in 2015 [26]. These four regioisomeric pairs (TB, TC, TD, and TE) of AAL-toxins can all induce genotype-specific necrosis characteristics in tomato leaflets in the same pattern as that of the TA pair, but they differ as much as 1000-fold in their relative toxicity [42].

## 2.4. Chemical and Structural Properties of Analogs of SAMs

In addition to fumonisins and AAL-toxins, several fungal secondary metabolites have also been identified as structural analogs of sphinganine and CerS inhibitors (summarized in Figure 3 and Table 1). These metabolites include myriocins, sphingofungins, viridiofungins, 2-amino-14,16-dimethyl-octadecan-3-ol (2-AOD-3-ol), and a new C17-SAM identified from mussels contaminated by marine fungi including *Aspergillus*, *Fusarium*, and *Trichoderma*. Australifungin, a structurally unrelated mycotoxin produced by *Sporormiella australis*, was also shown to inhibit sphingolipid synthesis in plants, similar to those of SAMs.

<figure><img src="image_158.png" /><figcaption>**Figure 3.** Chemical structures of other sphinganine-analog metabolites.</figcaption></figure>

Myriocins, sphingofungins, and viridiofungins inhibit serine palmitoyltransferase (SPT), while fumonisins, AAL-toxin, and australifungin inhibit sphinganine-N acyltransferase. Serine palmitoyl transferase, one of the key enzymes in the synthesis of sphingolipids, was also reported to play a positive role in PCD regulation. The increase of SPT activity promoted PCD in plants. In contrast, by inhibiting SPT activity, the excessive accumulation of sphingosine can be alleviated, leading to reduced PCD [43]. Therefore, myriocin are usually used as a SPT inhibitor to pretreat *Arabidopsis thaliana* and tomato plants to induce their resistance to FB₁ and AAL-toxin, respectively [44,45].
<table><caption>**Table 1.** Analogs of sphinganine-analog mycotoxins (SAMs), their fungal producer(s), and their activities.</caption><thead><tr><th>Analog of SAMs</th><th>Fungi/Origin</th><th>Activities</th><th>Scopus Citation (Review)</th><th>Reference</th></tr></thead><tbody><tr><td>Myriocins (thermoszymocidin, ISP-I)</td><td>Myriococcum albomyces Melanconis flavovirens Isaria sinclairii</td><td>Antifungal activity<br>Inhibitor of serine palmitoyltransferase (SPT)<br>Immunosuppressive activity<br>Protective effect on hepatotoxicity<br>Relieve fumonisin B<sub>1</sub> (FB<sub>1</sub>)-induced toxicity and cell death<br>Multi-pharmacological function on human</td><td>421(34)</td><td>[46–53]</td></tr><tr><td>Sphingofungins</td><td>E/F<br>A/B/C/D/I<br>G/H</td><td>Paecilomyces variotii Asp. fumigatus Asp. penicilliodes</td><td>Inhibitor of SPT<br>Antifungal activity</td><td>65(15)</td><td>[54–58]</td></tr><tr><td>Viridiofungins</td><td>A/B/C</td><td>Trichoderma viride Pers Tri. harzianum</td><td>Inhibitors of SPT and squalene synthase<br>Antifungal but lack antibacterial activity</td><td>21(5)</td><td>[59–61]</td></tr><tr><td>Australifungin</td><td></td><td>Sporormiella australis</td><td>Inhibitors of sphinganine-N acyl transferase<br>Antifungal activity, phytotoxicity</td><td>26(7)</td><td>[62,63]</td></tr><tr><td>2-AOD-3-ol</td><td></td><td>F. avenaceum</td><td>Animal cell toxicity as fumonisin B</td><td>5</td><td>[64]</td></tr><tr><td>C17-sphinganine analog mycotoxin</td><td>Contaminated mussels</td><td>Blocking skeletal muscle contraction</td><td>1</td><td>[65]</td></tr></tbody></table>
### 3. Relationships between SAMs' Structure and Biological Activities

The biological effects of SAMs, such as their toxicity, are similar among different SMAs. Many SAMs have a similar spectrum of susceptible plant species [34]. Tomato tissues and cells are similarly sensitive to AAL-toxins and to $FB_1$ and $FB_2$ toxins. In some other plants, AAL-toxins can cause necrotic cell death, similar to that of fumonisins [66]. For animal tissue cultures, the TA toxins can induce cytotoxicity in both rat liver and dog kidney cells as $FB_1$ toxin [67,68]. Besides, AAL-toxin and *F. verticillioides* could also inhibit larval growth and reduced pupal weights of tobacco budworm *Heliothis virescens* [69]. Such similarities have been attributed to the structural similarities between the SAMs and sphinganine. However, there are differences among SAMs in their biological effects and those differences are related to their structural differences. Below, we summarize the main findings in this area.

The amino functional group of SAMs is essential for their toxic activity. The peracetylated derivatives of AAL-toxins and $FB_1$ are biologically inactive or have significantly reduced toxicity in both the plant bioassay and the animal tissue culture systems [66,70,71]. These results were consistent with initial reports on these toxins showing that blocking the free primary amines of AAL-toxins by specific reagents could abolish the biological activities of these toxins in plants [72]. In an in vitro test of rat primary hepatocytes, it was noted that the N-acetyl analogue of $FB_1$, $FA_1$, also showed CerS inhibition [68]. Later, FA was found to spontaneously undergo isomerization, rearranging its O-acetylation group to form different analogs. The impact of these rearrangement products on inhibition of CerS in rat liver slices also supported the important role of a primary amino for both CerS inhibition and toxicity [73]. Derivatization of the amino group with fluorogenic reagents also makes the FBs' detection possible by the high-performance liquid chromatography (HPLC) assay [74]. FBs can bind covalently to proteins by reacting with amino groups in abiogenic conversions, which may increase the toxicity of those conversion products [75]. Similarly, the terminal amino group of $FB_1$ can conjugate to bovine serum albumin (BSA) and work as an immunogen to produce monoclonal antibodies for enzyme-linked immunosorbent assay (ELISA) detection [76]. Amino group of fumonisins can also work as an electron donor and react with the electrophilic carbon within the isothiocyanate (ITC) group. Consequently, FBs can be degraded by fumigation treatment with ITC-containing compounds [77].

The hydrolysis product of $FB_1$ ($HFB_1$) was shown as less toxic than both $FB_1$ and TA to plants [78]. Neither $HFB_1$ nor the yeast sphingolipids (completely acetylated) contain PTCA. While both had adverse effects on duckweed growth, they showed lower phytotoxicity than TA and $FB_1$ that contained one and two PTCA, respectively [79]. In contrast, the hydrolysis products of AAL-toxins largely maintain the toxicities of their parental compounds to the susceptible tomato lines [66]. These results indicate that PTCA is important to phytotoxicity of FBs and there is specificity of interaction between AAL-toxins and tomatoes.

Different from those in plants, an in vitro test using primary hepatocytes of rat showed that the FBs had greater cytotoxicity than FBs. However, the FBs could not initiate cancer development due to the lack of PTCA moiety, which was proposed to play an active role in the fumonisins absorption from the gut [70]. In the pregnant LM/Bc mouse model, $HFB_1$ did not cause neural tube defects. In contrast, 10 mg of $FB_1$/kg body weight of mice disrupted maternal sphingolipid metabolism, caused hepatic apoptosis in the female mice, increased fetus mortality, and reduced fetus weight [80]. In the SAMs-sensitive pig model, $HFB_1$ was shown to have limited intestinal or hepatic toxicity but only slightly disrupted sphingolipids metabolism [81]. The toxic effects of $FB_1$ and $HFB_1$ exposure on intestinal barrier function and immunity in a pig intestinal porcine epithelial cells and porcine peripheral blood mononuclear cells co-culture model was also investigated. $FB_1$ aggravated lipopolysaccharide (LPS)/deoxynivalenol (DON)-induced intestinal inflammation, while $HFB_1$ showed less toxicity to the immune system [82]. In addition, when $HFB_1$ and $HFB_2$ were acylated by CerS, the N-acyl-metabolites were toxic in vitro to the human colonic cell line and in vivo to the intraperitoneal rat tissues [83].

Fumonisins are capable of binding to polysaccharides and proteins via their two PTCA side chains in thermal-treated food and form fumonisin artifacts [84]. The activities of SAMs vary depending on where hydroxylation occurs along the carbon backbone. For example, $FB_2$ had a greater cytotoxic effect
than $FB_3$ and $FB_1$ in primary rat hepatocytes [70]. However, different from most other side groups, the C-1 terminal methyl group, which differed between FC and AAL-toxin from other fumonisins, seemed not required for the biological activity in SAMs.

Similar symptoms but less phytotoxicities of SAMs were observed when long-chain sphingoid bases or simple sphingolipids were applied to duckweed, which indicated that the phytotoxicity of SAMs might be resulted from the accumulation of phytotoxic sphingolipid intermediates [71,85]. This result was consistent with the induction of PCD through ceramide-based signaling pathways (described below).

Although AAL-toxins and fumonisins are structurally related chemicals with similar phytotoxicity, the latter are 10 times less efficient. AAL-toxins have been considered to serve as an herbicide at a very low dosage against a wide variety of broadleaf weeds (e.g., jimsonweed, prickly sida, and black nightshade). However, monocotyledonous crops (e.g., maize, wheat, and resistant varieties of tomato) are tolerant to AAL-toxins [24,86,87]. Until 2013, the mode of action through CerS inhibition was not among the 21 molecular target sites of the commonly used herbicides. Using AAL-toxin as a lead compound has the potential to develop novel and safe bioherbicide, which has phytotoxicity but reduced or no mammalian toxicity [88,89].

## 4. Detoxification of SAMs

Using agricultural and manufacturing practices for preventing the spread and growth of toxin-producing fungi and limiting mycotoxin production is the preferred method to eliminate food contamination by fungal toxins at the pre-harvest period [90]. However, it is extremely difficult to completely prevent fungal pathogen growth and mycotoxin contamination in agricultural practices and in food storage and processing. Since the toxicity of SAMs is structurally dependent, our knowledge on the relationships between SAMs' structure and biological activity provides clues for developing effective management strategies to minimize the impact of SAMs in food and feed products. Indeed, structural modifications such as hydrolysis have been demonstrated as effective at reducing the toxicity of SAMs [91].

Over the last three decades, chemical, biological, and physical strategies have been developed to degrade mycotoxins in food and feed products [92]. For example, nixtamalization was applied to reduce FBs by cooking fumonisin-contaminated maize with lime, as well as by using atmospheric ammoniation treatment [93–95]. Chlorine dioxide also showed the ability to degrade $FB_1$ [96]. Two common cooking methods include extrusion and nixtamalization were shown to reduce the toxicity of $FB_1$-contaminated corn [97]. Cold atmospheric pressure plasma was used as a physical treatment to successfully degrade pure $FB_1$ and AAL-toxins within 60 s, while the presence of the matrix slowed down the degradation [98,99]. Ozone was applied to disrupt fungal cells of *Fusarium* and *Aspergillus* by oxidizing sulfhydryl and amino acid groups of enzymes or attacking the polyunsaturated fatty acids of the cell wall [100]. However, not all SAMs are susceptible to physical and chemical treatments. In addition, some of these treatments may also result in derivatives with unknown toxicity and be detrimental for the treated commodities, as shown in some cases [101,102].

Another method to reduce SAM toxicity is through microbial actions. Microorganisms can carry out biotransformation reactions to detoxify SAMs [91]. Such methods include deamination, acetylation, hydrolysis, glucosylation, and decarboxylation. For example, Benedetti et al. isolated a Gram-negative rod bacterial strain from soil capable of degrading fumonisin to four metabolites when fumonisin was supplied as the sole carbon source [103]. The bacterium *Sphingopyxis* sp. could detoxify fumonisin B1 by at least two enzymatic steps, including an initial de-esterification reaction followed by de-amination of hydrolyzed product [104]. Chlebicz and Śliżewska found that 12 strains of *Lactobacillus* sp. bacteria and 6 strains of *Saccharomyces cerevisiae* yeast could reduce the concentration of $FB_1$ and $FB_2$ by 40% [105]. Similarly, Burgess demonstrated that fumonisin-producing *Asp. welwitschiae* have the ability to produce enzymes to synthesize non-aminated fumonisins that are less toxic than FB, and that those enzymes could be used for fumonisin detoxification [106]. Indeed, using enzymes to detoxify by
modification of chemical structures has become a promising method for mycotoxins control after grains harvest [107,108]. For example, fumD (carboxylesterase) from *Sphingopyxis* catalyze detoxification of FB₁ to the hydrolyzed form by hydrolysis of both PTCA side chains. Then, the aminotransferase FumI could degrade FB₁ by catalyzing the deamination of HFB₁. FumD has also been tested for interference of fumonisins adsorption in turkey, swine, and pig [109–111]. Finally, several other enzymes such as manganese peroxidase from lignocellulose-degrading fungi and laccase from *Pleurotus eryngii* were all capable of degrading fumonisins [112,113].

Another potential method to reduce SAMs from food and feed products is to use adsorbent materials to soak up and remove the toxins. Many materials have shown the capacity to adsorb mycotoxins in vitro, thus the use of adsorbents in livestock diet as feed additives can potentially decrease the bioavailability of mycotoxins to humans and animals. As feed additives, cholestyramine, nanosilicate clay platelets, and refined calcium montmorillonite clay all reduced FB₁ toxicosis [114–116]. Moreover, natural products such as the phenolic compound chlorophorin, honey, and cinnamon oil have all shown promise as fumonisin-reducing agents [117–119].

## 5. Programmed Cell Death and Sphingolipids

Almost all cells die eventually. There are four main types of cell death: necroptosis, pyroptosis, ferroptosis, and apoptosis, classified based on their distinct molecular and cellular processes and different outcomes. Apoptosis or programmed cell death (PCD) is a kind of cell suicide that strictly regulates cells that are no longer needed or are a threat to the wellbeing of multicellular organisms. Both plants and animals have PCD and they are functionally analogous to each other [120–123]. PCD plays essential roles to maintain normal physiological activities in multicellular organisms such as plants and animals and is an active self-regulating process to selectively eliminate redundant, aged, and damaged cells. PCD can be predicted for specific cells at defined developmental stages. However, PCD can also be induced by membrane-bound and cytosolic proteins stimulated by stress-induced signals. Such signals can trigger cell death via intricate cascades of transcriptional changes and post-translational protein modifications [122,124]. The characteristics of PCD include reduced cell volume, chromatin marginalization and condensation, nuclear lamina disassembly, DNA fragmentation, and apoptotic body formation, etc. [1].

PCD triggers and propagation involve many factors, including the expressions of certain cell surface receptors, transmembrane domains of several membrane proteins, intracellular proteins related to the propagation of death signals, secondary messengers including inositol triphosphate and ceramides, calcium (Ca²⁺) fluxes, reactive oxygen species (ROS), regulatory factors of cell cycle, and other suppressors or activators proteins. Many of these subcellular components, genes, and signal transduction pathways involved in PCD are functionally conserved across all domains of cellular organisms, from bacteria to fungi to plants and animals. However, there are differences in the actual mechanisms among organisms, as summarized in References [125,126].

Sphingolipids have been implicated to play an important role in cell growth, development, response to external environment, and PCD. As the main component of the cell membrane system, sphingolipids help to maintain the structural stability and transport of molecules across cell membranes [127,128]. In mammals, sphingolipids are especially abundant in the nervous system cells, with important functions in cell contact, growth, differentiation, communication, response to stress signals, and apoptosis [30,129]. In plants, sphingolipids are involved in response to both biotic and abiotic stresses, such as to pathogen infection, drought, and low temperature [31,130,131]. Indeed, the linkage of ceramide signaling to apoptosis has been widely reported in both plants and animals. Consequently, actions by SAMs to disrupt the functions of sphingolipids could have significant negative consequences. However, our knowledge about the roles of sphingolipids on apoptosis have also led to increasing interests on potential novel therapies using sphingolipids as treatment targets against degenerative and proliferative diseases in humans and animals, such as cancer and Parkinson's disease [132,133].
A large number of studies have shown that sphingolipids could serve as critical secondary messengers in signal transduction to regulate PCD [134,135]. For example, in neutrophils, sphingolipids have been linked to increased superoxide formation and Ca²⁺ influx, which are universal signaling molecules involved in many cellular functions [136]. The induction of PCD in *Arabidopsis* by ceramides was also verified to be partly dependent on ROS in mitochondria or regulated by the release of Ca²⁺ [137,138]. In addition, increase of sphingosine level can activate the mitogen-activated protein kinase (MAPK) pathway in which MPK6 participates, promote the accumulation of sialic acid (SA), and then induce PCD [43]. Lachaud et al. found that the activated calcium-dependent kinase (CPK3) can regulate the process of sphingosine-induced PCD by dissociating CPK3 from the 14-3-3 protein-complex under increased calcium concentration induced by sphingosine. The activated CPK3 is then degraded followed by PCD induction [139].

Mutational studies of *Arabidopsis* and in vitro experiments have shown that ceramides, and free sphingoid bases such as sphingosine, sphinganine, and phytosphingosine, can all induce PCD. In contrast, phosphorylated products of these compounds can inhibit or alleviate PCD in plants [44,138,140–142]. However, phosphorylated sphingosine can inhibit the growth of yeast cells, which suggests that there are different mechanisms of action between plants and yeasts [143]. It is worth mentioning that not all ceramides can induce PCD. Nagano et al. found that when the C2 position of fatty acid in the side chain of ceramides was hydroxylated, PCD was inhibited rather than induced [144]. These results indicate that the occurrence of PCD in plants depends not only on the absolute content of sphingolipids, but also the relative ratios of various modified forms. In the next section, we will describe how SAMs are involved in PCD.

## 6. SAMs Trigger PCD through Ceramide-Based Signaling Pathways

In plants, pathogen invasions can lead to disruptions in host cellular homeostasis, and trigger cell death in susceptible varieties or even in resistant varieties with a hypersensitive response (HR). Because of its many similarities with PCD, HR is often considered as a form of PCD in plants. AAL-toxins, as the pathogenic factor of tomato stem canker disease, can induce PCD in sensitive tomato varieties resulting in fragmentation of chromosomal DNA and formation of apoptotic bodies in cells [145]. Similarly, when treated by FB₁ produced by pathogenic *Fusarium*, *Arabidopsis* protoplasts showed symptoms similar to PCD in animal cells [146]. At the tissue and organ levels, *Arabidopsis* leaves treated with the FB₁ toxin showed characteristic disease symptoms. Cells of the diseased leaves had overall phenotypes similar to HR, including callose accumulation, ROS production, and pathogenesis-related (PR) gene induction [147]. The damages caused by SAMs on host plants can further increase pathogen infection and colonization. Similar to those found in plants, SAMs can induce neuro-/renal-responses, heptatoxicosis, and neoplasms, as well as cell death in animals. The relationship between apoptosis and ceramide signaling has been established in both plants and animals in their response to SAMs [120]. For example, the induction of cell death in both tomato and African green monkey kidney (CV-1) cells occurred under similar toxin concentrations and time frames. For both types of cells, morphological markers characteristic of apoptosis were observed, including cells with positive terminal deoxynucleotidyl transferase end labeling (TUNEL), DNA fragmentations, and the formation of apoptotic-like bodies [145,148].

SAMs are structurally analogous to sphinganine and are thus effective inducers of PCD. The emerging mechanism of their actions is that SAMs can competitively bind to CerS in cells. Such binding leads to the accumulation of free sphingoid bases, the substrates of CerS, while ceramides as products of CerS were consumed and reduced, activating PCD in plant and animal cell lines [3,85]. For example, it was reported that FB₁ is a potent competitive inhibitor of CerS from liver and brain microsomes in several mammalian cell lines [149,150]. An increase in sphinganine was observed in an in vivo test of CerS inhibition, as well as in FB-fed animals treated at high concentrations [2]. In addition, TA and FB₁ can inhibit CerS in rat hepatocytes and green tomato fruits [150,151]. Furthermore, it was found that FB₁ not only induced apoptosis in animal cells, but also altered cell morphology, cell–cell
interactions, cell surface proteins behavior, protein kinase activity, and cell growth and viability in non-apoptotic cells [148,152]. In plant cells, after exposure to SAMs, sphingosine concentration increased significantly within a short time, followed by the accumulation of ROS in the cytoplasm and then apoptosis. These results suggested that the accumulation of sphingosine in cells was the upstream signal of ROS for cell death [43,44,85]. The induction of PCD by $FB_1$ is also related to the accumulation of ceramides. In *A. thaliana*, there are two types of CerSs that use different substrates. Class I CerSs use sphinganine and C-16 fatty acyl-CoA as substrates, while class II use phytosphingosine and very long-chain fatty acyl-CoA as substrates. $FB_1$ mainly inhibits the activity of class II CerSs. When treated with $FB_1$, phytosphingosine in cells increases significantly. At the same time, as the product of a previous step, sphinganine also increases, which provides more substrates for class I CerS. Consequently, the products of class I CerS in cells increase, leading to induced PCD [153,154].

## 7. Plant Resistance to SAMs

Phytohormones are also involved in the defense reaction induced by SAMs. Changes in ethylene (ET) were first discovered in AAL-induced necrosis of tomato [155]. Alteration in ethylene perception in "never ripe" mutants of tomatoes can markedly alleviate the tissue damage caused by SAMs, which indicated an ethylene-associated signal transduction during plant cell death [156]. Later, a transcription analysis of AAL-toxin-induced cell death was carried out in *Arabidopsis*. Genes responsive to ROS and ET were among the earliest upregulated genes [157]. Mase used VIGS (virus-induced gene silencing) analyses and verified that the ET signaling pathway and MAPK cascades were required for AAL-toxin-induced PCD in tobacco [158]. By SA-mediated ET suppression, glutathione (GSH) may be involved in resistance primarily against AAL-toxin-induced stress in Arabidopsis [159].

Unlike ethylene in host basal defense responses against the tomato pathotype of *A. alternata*, the jasmonate (JA)-dependent signaling pathway is not involved in host defense against the toxigenic *A. alternata* pathogen. JA affects pathogen acceptability via a toxin-independent mannerin in the interactions between plants and toxigenic necrotrophic fungal pathogens. It may act upstream of ethylene biosynthesis in AAL-toxin-triggered tomato cell death [160,161]. Later, a comparative proteomics analysis revealed that the COI1 (coronatine insensitive 1, JA receptor)-dependent JA pathway enhances AAL-toxin-induced PCD of tomato through regulating the redox status of the leaves, other phytohormone pathways, and/or important PCD components [162].

The sensitivity of tomato plants to the fungal pathogen *A. alternata* f. sp. lycopersici is controlled by the *Alternaria* stem canker resistance locus (*Asc*-locus) on chromosome 3 [163]. Mutations of tomato *Asc* locus gives resistance to the pathogen, while overexpression of the tomato *Asc-1* gene mediates high insensitivity to SAMs in tomato and confers resistance to pathogen infection in sensitive *Nicotiana* plants [164,165]. *Asc-1* is a homolog of the yeast longevity assurance gene *LAG1*, which encodes components of sphinganine N-acyltransferase. This resistance gene could prevent the disruption of sphingolipid metabolism during AAL-toxin-induced PCD. Both *Nicotiana* and *Lycopersicon* genera belong to Solanaceae. In tomato, insensitivity to SAMs and susceptibility to the pathogen is determined by *Asc-1* [166]. In contrast, the SAM-sensitive species in the *Nicotiana* (except for *N. umbratica*) still have *Asc-1* homologs and are resistant to *A. alternata* f. sp. lycopersici infection with HR, which indicates an additional (non-host) resistance mechanism between *Nicotiana* and this pathogen [167]. The multilayered defense systems also exist in *Arabidopsis* non-host resistance to *A. alternata* [168]. Similarly, although many *Fusarium* species produce fumonisin, they cannot infect AAL-sensitive tomato. This non-host resistance includes a multi-layer defense system involving both pre- and post-invasion, and help plants defend against various pathogens [169]. In addition, Zélicourt demonstrated that two of three *Lag1* homologs in the *Orobanche cumana* genome were responsible for an enhanced sensitivity to AAL-toxin [170].

Aside from the above-mentioned genes, several other genes were identified from Arabidopsis and found to be involved in the AAL-induced PCD pathway, including Zinc *A. thaliana* 11 (a zinc finger protein ZAT11), fbr41 ($FB_1$ Resistant41), and baculovirus p35 gene (inhibitor of a class of cysteine
proteases). All of them showed protective effects on AAL-toxin-induced cell death and pathogen infection in plants [171–173]. Discovery of resistant genes has provided a potential strategy for SAMs’ control in crop production by plant transgenic modification.

Because of the high toxicity of fuminisins, especially FB₁, a large number of studies have focused on them. So far, the mechanism of FB₁ toxicity has been centered around its structural resemblance with sphinganine and consequent competitive inhibition of CerS and the disruption of lipidomic profiles. However, there is emerging evidence suggesting that FB₁ can disrupt mitochondrial function and generate excessive toxic ROS.

Table 2 shows a list of reviews summarizing the latest advances related to fumonisins, in their assessment, biosynthesis, detection, crop breeding of resistant varieties, and toxicity.

<table><caption>**Table 2.** Topical reviews on fumonisins over the last five years.</caption><thead><tr><td>Subject</td><td>Content</td><td>Reference</td></tr></thead><tbody><tr><td>Assessment</td><td>Biomarkers, metabolism, and biomonitoring of fumonisins in human biological fluids</td><td>[174]</td></tr><tr><td>Assessment</td><td>Impact on agriculture, food, and human health and their management strategies</td><td>[16]</td></tr><tr><td>Assessment</td><td>Risk assessment and intervention models for fumonisin of maize in South Africa</td><td>[175]</td></tr><tr><td>Assessment</td><td>Fumonisins and related <i>Fusarium</i> occurrence in wheat and its by-products</td><td>[176]</td></tr><tr><td>Assessment</td><td>Fumonisins and their modified forms</td><td>[177]</td></tr><tr><td>Assessment</td><td>Biological methods for fumonisins reduction and related <i>Fusarium</i> species control</td><td>[178]</td></tr><tr><td>Assessment</td><td>Fumonisins and <i>A. alternata</i> f. sp. <i>Lycopersici</i> (AAL) toxins in ruminants and their forages</td><td>[19]</td></tr><tr><td>Biosynthesis</td><td>Genetic regulation of fumonisins biosynthesis by specific genes and global regulators</td><td>[179]</td></tr><tr><td>Biosynthesis</td><td>Impact of environmental variables and genetics of maize resistance on fumonisin accumulation</td><td>[180]</td></tr><tr><td>Detection</td><td>Analytical methods for fumonisins detection in single corn kernels</td><td>[181]</td></tr><tr><td>Detection</td><td>Molecular methods for early detection of fumonisin-producing <i>F. verticillioides</i></td><td>[182]</td></tr><tr><td>Plant resistant</td><td>Genomic, genes, and pathways in maize resistance to <i>Fusarium</i> ear rot and fumonisin accumulation</td><td>[183]</td></tr><tr><td>Plant resistant</td><td>Relationship between Bt maize hybrids and fumonisins contamination level</td><td>[184]</td></tr><tr><td>Toxicity</td><td>Mitochondrial toxicity induced by FB<sub>1</sub></td><td>[185]</td></tr><tr><td>Toxicity</td><td>Molecular mechanisms underlying FB<sub>1</sub>-mediated toxicities and related interventions</td><td>[186]</td></tr><tr><td>Toxicity</td><td>CerS inhibition by fumonisins result in animal and plant disease</td><td>[187]</td></tr><tr><td>Toxicity</td><td>Dietary fumonisin and growth impairment in children and animals</td><td>[188]</td></tr><tr><td>Toxicity</td><td>Impact of fumonisin-contaminated feed on pig intestinal health</td><td>[189]</td></tr><tr><td>Toxicity</td><td>Oxidative stress-mediated toxicity and metabolism in vivo and in vitro</td><td>[190]</td></tr></tbody></table>

## 8. Genes Responsible for SAMs Production

In fungi, genes directly involved in the biosynthesis pathway of the same secondary metabolite are usually located at adjacent positions in gene clusters in the genome [191]. These genes are often co-expressed and co-regulated. The genes involved in fumonisin biosynthesis fit this general pattern. Specifically, *Fusarium* species capable of producing fumonisins typically contain one gene cluster involved in their synthesis, called the *FUM* gene cluster. At present, a total of 21 genes have
been identified in *FUM* gene clusters of various species and verified to be involved in fumonisin biosynthesis and regulation or self-protection using a variety of approaches, such as gene knockouts, domain swapping, and heterologous expression (Table 4) [192–197]. In *F. verticillioides*, the *FUM* cluster responsible for fumonisin B biosynthesis includes 17 genes [193,197,198].

As described previously, SAMs are polyketide-derived compounds with structural similarity to sphinganine. Polyketides are synthesized by polyketide synthases (PKSs), which are large multifunctional enzymes. *FUM1* is a PKS gene previously designated as *FUM5* [199]. PKS encoded by *FUM1* catalyzes the synthesis of an octadecanoic acid precursor as the initial step for FB biosynthesis in *Fusarium* spp. [200,201]. The proposed biosynthetic pathway of FB is described in Figure 4 [197]. The precursor mentioned above undergoes condensation with L-alanine to synthesize the polyketide backbone, this reaction was catalyzed by the aminotransferase Fum8 [192,201]. In *F. oxysporum* strain O-1890, the orthologue of *FUM8* determines that *Fusarium* produces predominantly FCs [195]. The *fum8* deletion in some stains of *A. welwitschiae* is also considered to be associated with the loss of FB<sub>2</sub> production [202].

<figure><img src="image_159.png" /><figcaption>**Figure 4.** The *FUM* gene cluster, the proposed pathway of fumonisin biosynthesis, and the proposed mechanism for self-protection against fumonisins toxicity by the toxin-producing fungi (FBs and FCs). SAM, S-adenosyl methionine; VE, vesicles; MT, mitochondrion; ER, endoplasmic reticulum; NU, nucleus; PM, plasma membrane.</figcaption></figure>

A likely mitochondrial carrier protein encoded by *FUM11* transport the substrate tricarboxylate for Fum7 (dehydrogenase) and Fum10 (acyl-CoA synthase) to produce CoA-activated
tricarballylic acid, which are attached to the polyketide backbone by Fum14 (condensation-domain protein) [192,194]. The ensuring steps of fumonisin biosynthesis involving various modifications of the backbone (including primarily hydroxylation) were catalyzed by several enzymes, such as Fum6/Fum12/Fum15 (cytochrome P450 monooxygenase), Fum2 (hydroxylase of C10), Fum13 (short-chain dehydrogenase/3-ketoreduction), and finally, Fum3 (hydroxylase of C5, dioxygenase), catalyzed by FUM9-encoded protein (alleles of FUM3) [192,193,203–206].

In addition, FUM21 encoding a GAL4-like Zn(II)2Cys6 transcription factor was verified to be involved in the regulation of fumonisin synthesis. However, it seemed that the deletion of FUM16 had no apparent effect on fumonisin production in F. verticillioides [194,198,207–209]. Recently, FUM17–FUM19 in F. verticillioides were found to help the fungus to avoid its own toxicity during fumonisin production. Fum19 is an ATP-binding cassette transporter (ABC transporter) and acts as a repressor of the FUM gene cluster. FUM17 and FUM18 are CerS homologs. FUM18 could fully complement the yeast CerS null mutant LAG1/LAC1, while co-expression of FUM17 and CER3 partially complemented. Both the Fum17 and Fum18 proteins enable F. verticillioides to increase its resistance of fumonisin by providing FUM cluster-encoded CerS activity as a first level of self-protection [197].

Aside from the FUM genes, other genes like FST1 (transporter), FUG1 (transcription or signal transduction factors), CPP1 (protein phosphatase type 2A catalytic subunit), and FvVE1 (regulator) in F. verticillioides, PKS3 and PKS11 in F. proliferatum, and GATA-type transcription factors AreA and AreB (known as the global nitrogen regulators) in F. fujikuroi have also been demonstrated to have an important role in fumonisin biosynthesis and regulation [209–214]. In addition, a degenerated, over-represented motif which is potentially involved in the cis-regulation of FUM genes and fumonisin biosynthesis was also identified from both F. verticillioides and Asp. niger, while it was not found in fumonisins non-producing fungi containing various FUM homologues [215].

Several abiotic and biotic factors have been found to affect the expression of FUM genes and regulate biosynthesis of fumonisin. These factors include water activity, temperature, carbon sources and other nutrients, host plant species and varieties or their extracts, and plant age [216–220]. Mature plants and extracts from those plants are often associated with higher concentrations of SAMs. It has been suggested that harvesting the crop at earlier stages other than full maturity could be one of the strategies to control fumonisin contamination [221,222].

The genome sequencing and analysis of Asp. niger revealed that its genome contained a gene cluster (fum cluster) homologous to the FUM cluster in Fusarium species (shown in Table 4). Specifically, 12 homologues of the fumonisin synthesis genes were found, including fum1, fum3, fum6, fum7, fum8, fum10, fum13 to fum16, fum19, and fum21 genes [208,223,224]. This gene cluster is also found in fumonisin-producing isolates of Asp. welwitschiae but is absent from the genomes of other sequenced Aspergilli that do not produce fumonisin, such as Asp. fumigatus, Asp. oryzae, and Asp. nidulans [7,208]. In addition, homologs of multiple fum genes have been found in several other Aspergilli spp. but where no fumonisin production has been detected (summarized in Table 3). Some of the Aspergullus spp. contain genes that are unique to them. For example, a dehydrogenase gene (sdr1) of a short-chain length was found in the fum cluster of Asp. niger but is absent in the FUM gene cluster of Fusarium spp. In contrast, the Fusarium FUM2 gene with a function of hydroxylation at the C-10 backbone position of fumonisin is absent in the Asp. niger fum cluster [207,208]. This result is consistent with the study that shows that Asp. niger only produces fumonisins FB2, FB4, and FB6, which lack a hydroxyl at C-10 [7,225,226]. However, isolates of several black aspergilli (including A. niger, Asp. foetidus, and A. tubingensis) isolated from peanuts and maize also produced FB1 and FB3, consistent with a complex biosynthesis pattern of the fumonisins in Aspergilli spp. [227].
<table><caption>**Table 3.** Difference in genomic context of fumonisin biosynthetic gene (*fum*) cluster between strains of *Aspergillus* spp.</caption><thead><tr><th>Fungi</th><th>Stains</th><th>fum Cluster</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">Asp. niger</td><td>fumonisin-producing strains</td><td>fum cluster</td><td>[208]</td></tr><tr><td>fumonisin-non-producing strains</td><td>Intact fum cluster</td><td>[7,235]</td></tr><tr><td rowspan="2">Asp. welwitschiae</td><td>fumonisin-producing strains</td><td>fum cluster</td><td>[7]</td></tr><tr><td>fumonisin-non-producing strains</td><td>Three fum cluster types including an intact cluster</td><td>[7,235]</td></tr><tr><td rowspan="2">Asp. tubingensis</td><td>fumonisin-producing strains</td><td>Not tested</td><td>[227]</td></tr><tr><td>fumonisin-non-producing strains</td><td>Multiple patterns of fum gene deletion</td><td>[7]</td></tr><tr><td>Asp. brasiliensis</td><td>fumonisin-non-producing strains</td><td>Multiple patterns of fum gene deletion</td><td>[7]</td></tr><tr><td>Asp. luchuensis</td><td>fumonisin-non-producing strains</td><td>Multiple patterns of fum gene deletion</td><td>[7,236]</td></tr><tr><td>Asp. fumigatus</td><td>fumonisin-non-producing strains</td><td>Not detected</td><td>[208]</td></tr><tr><td>Asp. oryzae</td><td>fumonisin-non-producing strains</td><td>Not detected</td><td>[208]</td></tr><tr><td>Asp. nidulans</td><td>fumonisin-non-producing strains</td><td>Not detected</td><td>[208]</td></tr><tr><td>Asp. foetidus</td><td>fumonisin-producing strains</td><td>Not tested</td><td>[227]</td></tr></tbody></table>
<table><caption>**Table 4.** Homologous genes and their functional roles in the biosynthesis of SAMs.</caption><thead><tr><th colspan="2">Homologue of Cluster Genes</th><th>Predict Gene Product and Function</th><th>Reference</th></tr></thead><tbody><tr><td>fumonisin</td><td></td><td>AAL-toxin</td><td></td></tr><tr><td>Fusarium spp.</td><td>Aspergillus spp.</td><td>A. alternata</td><td>[169,224]</td></tr><tr><td>FUM1<br>(fumonisin biosynthetic gene 1,<br>previously designated as FUM5))</td><td>fum1</td><td>ALT1<br>(AAL-toxin biosynthetic<br>gene 1)</td><td>polyketide synthase<br>[12,199,200]</td></tr><tr><td>FUM2</td><td>absent</td><td></td><td>Dioxygenase for hydroxylation of C10<br>[228]</td></tr><tr><td>FUM3</td><td>fum3</td><td></td><td>Dioxygenase for hydroxylation of C5 (the same gene as FUM9)<br>[205,228,229]</td></tr><tr><td>FUM4</td><td></td><td></td><td>Not clear<br>[230]</td></tr><tr><td>FUM6</td><td>fum6</td><td>ALT2</td><td>Cytochrome P450 monooxygenase-reductase fusion proteins for<br>hydroxylation of C14/C15<br>[192,231]</td></tr><tr><td>FUM7</td><td>fum7</td><td>ALT3</td><td>Type III alcohol dehydrogenases for PTCA<br>(propane-1,2,3-tricarboxylic acid) side chain formation<br>[192,194,232]</td></tr><tr><td>FUM8</td><td>fum8</td><td></td><td>α-oxoamine synthase and homologous for amino transfer and<br>FBs/FCs production<br>[192,195]</td></tr><tr><td>FUM10</td><td>fum10</td><td></td><td>Fatty acyl-CoA synthase for PTCA esterification<br>[193,194]</td></tr><tr><td>FUM11</td><td></td><td></td><td>mitochondrial transport protein for PTCA transport<br>[193,194]</td></tr><tr><td>FUM12</td><td></td><td></td><td>cytochrome P450 monooxygenases<br>[193]</td></tr><tr><td>FUM13</td><td>fum13</td><td>ALT6</td><td>Short-chain dehydrogenase/ketoreductase of C3<br>[193,204,206]</td></tr><tr><td>FUM14</td><td>fum14</td><td></td><td>Non-ribosomal peptide synthetase for PTCA esterification<br>[193,194,233]</td></tr><tr><td>FUM15</td><td>fum15</td><td></td><td>Cytochrome P450 monooxygenases<br>[193]</td></tr><tr><td>FUM16</td><td>fum16</td><td></td><td>Fatty acyl-CoA synthetase<br>[193,194,224]</td></tr><tr><td>FUM17</td><td></td><td></td><td>CerS for self-protection against fumonisins<br>[193,197]</td></tr><tr><td>FUM18</td><td></td><td></td><td>CerS for self-protection against fumonisins<br>[193,197]</td></tr><tr><td>FUM19</td><td>fum19</td><td></td><td>ABC transport protein as a repressor of FUM gene cluster<br>[193,197]</td></tr><tr><td>FUM20</td><td></td><td></td><td>Not clear<br>[234]</td></tr><tr><td>FUM21</td><td>fum21</td><td>ALT13</td><td>Zn(II)2Cys6 transcription factor<br>[198]</td></tr><tr><td>absent</td><td>SDR1</td><td></td><td>Short-chain dehydrogenase/reductase (SDR)<br>[7]</td></tr></tbody></table>
AAL-toxins are produced by *A. alternata* f. sp. *Lycopersici*, a specific pathotype of a common plant fungal pathogen in a genus different from *Aspergillus* and *Fusarium*. This pathotype can produce polyketide-derived compounds similar in structure to fumonisins produced by *Fusarium* species. The *ALT* (AAL-toxins synthesis) genes are also located as a cluster on a conditional disposable (CD) chromosome of ~1.0 Mb in all strains of the tomato pathotype of *A. alternata* from different countries [237]. Such CD chromosomes carrying a toxin biosynthesis gene cluster were also found in other pathotypes of *A. alternata* [169]. They control other HSTs production and pathogenicity to their host. They could maintain stably in a new genetic background to an expanded range of pathogenicity, which was verified by a protoplast fusion test [238]. The AAL-toxin gene cluster includes at least 13 genes in a 120 kb region, some of which showed significant similarity to the *FUM* gene cluster consisting of 17 genes in a 45.5 kb region. However, the arrangement of genes in the *ALT* and *FUM* clusters differs between these two groups of fungi. In addition, in one strain, As-27 of *A. alternata*, there were two sets of the AAL-toxin biosynthetic gene cluster on the CD chromosome [169]. The synthesis of AAL-toxins was found to be initiated by *ALT1*-encoded PKSs to produce the aminopentol backbone, which was then modified by other enzymes [239]. The functional similarity between *ALT1* and *FUM1* was confirmed when fumonisin biosynthesis in *FUM1*-disrupted *F. verticillioides* was restored when complemented by the *ALT1* from *A. alternata* [240]. Similarly, expression of *ALT1* and production of AAL-toxins were also found to be regulated by the global regulator *LaeA* [241]. AAL-toxin accumulation also benefits from high water activity (0.995 aw) and high temperature (above 30 °C) during the incubation period of the pathogen [242].

Interestingly, a PKS gene similar to *FUM1* and orthologs of the *FUM* gene cluster were found in the genome of *Cochliobolus* spp. by phylogenetic analysis of fungal polyketide. These fungi were also speculated to produce a fumonisin or other SAMs [243,244]. To predict the potential distribution of SAM production in fungi, Kim et al. proposed a hypothesis on SAM biosynthetic gene clusters based on fumonisin biosynthesis model. This putative gene cluster should include a PKS, an aminotransferase, and a dehydrogenase gene. Their model showed that sixty-nine species of the *Fusarium* genus and species of twenty-four other fungal genera were predicted to have at least one SAM cluster [245].

## 9. Evolution of SAMs Production

Horizontal gene transfer (HGT) has been proposed as a major mechanism responsible for the acquisition and evolution of fumonisins and AAL-toxins biosynthetic gene clusters among divergent fungi [7,9,169,224]. In *Fusarium*, genome sequence analyses revealed that the fumonisin biosynthetic genes (*FUM*) are clustered and show a consistent gene organization among most species. For example, the *FUM* clusters in *F. oxysporum*, *F. proliferatum*, and *F. verticillioides* exhibit relatively little variability, with the order and orientation of genes within the clusters all being the same as each other. In addition, their sequence variability among the orthologues of coding regions from *F. oxysporum* and *F. verticillioides* is relatively low [193,195]. The two different species of *Aspergillus* that produce fumonisins, *Asp. niger* and *Asp. welwitschiae*, are also similar to each other in their gene order but different from that of the *FUM* cluster in *Fusarium* [224]. At present, the tomato pathotype of *A. alternata* was the only species of genus *Alternaria* capable of producing SAMs and this pathotype has clustered genes (involved in AAL-toxin biosynthesis) similar to the *FUM* cluster in *Fusarium* [169,237]. Together, the gene structure and sequence analyses suggested that the SAMs biosynthetic gene cluster likely originated in *Fusarium* and transferred to *Asp. niger* and *A. alternata* by HGT. The similarities in chemical structure and cytotoxicity on plants and animals between fumonisins and AAL-toxins are also supportive of this hypothesis. However, the differences between *FUM* clusters and the AAL-toxins biosynthetic gene cluster also suggested that there has been significant divergence between them.

Analyses of the *FUM* gene cluster among *Fusarium* species also revealed evidence for gene gain, loss, and mutations of different genes. For example, not all *Fusarium* species can produce fumonisins. Even for species that can synthesize fumonisins, some strains produce more than others under the same experimental conditions, while other strains do not produce the toxins at all [9,196]. Indeed, for certain
strains, while the *FUM* genes were detected, there was no detectable fumonisin. This was likely
due to the accumulation of mutations leading to the *FUM* genes being nonfunctional. For example,
several mutations have been found in *FUM7* and *FUM21* in *F. fujikuroi* [246]. Furthermore, several
fumonisin-non-producing *Fusarium* species lack the fumonisin biosynthetic genes but retain homologs
of several genes that flank the Fum cluster in *F. verticillioides* [247]. Interestingly, the flanking regions
of the *FUM* cluster often differ between species, consistent with the independent origins of the *FUM*
cluster, including independent acquisition and/or loss of the gene cluster by fumonisin-producing
species [195]. For example, fumonisin-non-producing strains of *F. verticillioides* isolated from banana
did not contain the functional fumonisin biosynthetic gene (*FUM*) cluster but did contain portions of
*FUM21* and *FUM19* flanking the cluster, both of which are the terminal genes at each end of the *FUM*
cluster. However, the banana strains are still pathogenic to banana, but they do not show the same
pathology as the fumonisin-producing strains do on maize. When a banana strain was co-transformed
with two overlapping cosmids containing the entire *FUM* gene cluster, fumonisin production and
pathogenicity on maize seedlings were recovered [248]. Similar to *Fusarium*, FB-non-producing
isolates of *Asp. niger* or other *Aspergillus* species also had an intact *fum* cluster or multiple patterns
of *fum* gene deletion, respectively (shown in Table 3). Similarly, the AAL-toxin-production gene
cluster in *A. alternata* was likely derived from *Fusarium* species by an HGT event. Evidence from the
*ALT* gene cluster distributed in isolates of *A. alternata* also supports this hypothesis of HGTs within
AAL-toxin-producing pathogens [237]. Together, these results suggest that there have been multiple
HGTs of the cluster between species, as well as duplication and loss of the whole or part of the cluster
after acquisition [9].

The hypothesis that multiple HGTs were involved in generating the current distribution of *FUM*
gene clusters is further supported by phylogenetic studies. Specifically, phylogenetic trees based on
genes from the *FUM* cluster often do not parallel that of the *Fusarium* species tree based on other
genes [9]. In *Fusarium*, the translation elongation factor (tef-1α) gene is the most commonly used marker
gene for taxonomic studies. However, sequences of the tef-1α are often insufficient for distinguishing
fumonisin-producing isolates from different countries and/or host plants. In contrast, DNA sequence
polymorphisms based on *FUM1* often provide better resolutions among pathotypes [196,249–253].
For example, phylogenetic analysis of 38 *F. proliferatum* isolates originating from different hosts showed
that sequence variation among strains in the *FUM1* gene was correlated with that of the host plants.
Specifically, phylogenetic analysis based on the partial *FUM1* sequences differentiated the host-related
groups more clearly than that based on tef-1α sequences. The best distinguished group consists of
garlic-derived isolates and formed a separate branch on a *FUM1*-based dendrogram [196,254]. Similarly,
*FUM1* sequence divergence analysis on *F. proliferatum* and *F. verticillioides* strains isolated from pea also
formed a distinct group when compared to strains derived from different host species [253].

Aside from gene differences among isolates from different host plants, variations of both toxigenic
potential and growth patterns may also differ between isolates derived from the same host plants.
While no difference was observed in FB levels measured among pea seeds, the FB productions differed
between selected strains of *F. proliferatum* in rice cultures [253]. Among all these test isolates of
*F. proliferatum*, the most varied group of isolates found so far were those isolated from maize [196,254].
Both the inter- and intra-specific variation in FBs synthesis level can at least partly be explained by the
sequence differences inside the FUM cluster.

In summary, the analyses so far suggested that the *FUM* gene cluster was responsible for fumonisin
biosynthesis. Mutation and deletion of some or all of the genes in the cluster could result in limited
or no production of fumonisin, leading to a weaker disease development of the pathogen on the
host plant. Sequence analyses showed that *A. alternata* has likely gained the ability for AAL-toxin
production due to HGT of the SAMs gene cluster from fumonisin-producing *Fusarium* species followed
by independent evolution in pathogen–host interaction. The divergent patterns of toxin biosynthesis
gene sequence divergence may explain the differences between fumonisins and AAL-toxins in both
their productions and their impacts on host–pathogen interactions.
## 10. Detection Method of SAMs

A variety of methods have been developed to detect SAMs, including HPLC with fluorescence/evaporative light scattering detection or mass spectrometry (MS), thin-layer chromatography (TLC), ELISA, Fourier transform near infrared (FT-NIR) spectroscopy, and so on [255-263]. While these traditional analytical methods were designed to detect and quantify known compounds for which standards are available, there is clear evidence that many unknown derivatives may exist in food and food products and some of these could be toxic to animals, including humans [39]. In 2015, a semi-targeted method combining product ion filtering and rapid polarity switching was designed for fast detection of all known fumonisins and AAL-toxins. Some new structurally related emerging toxins were also discovered by this method [26].

Aside from method development that targets potentially novel SAMs not reported before, there are also developments for efficient methods that target the detection of known SAMs. Indeed, several new methods based on immunoassay were developed for simple, rapid, and ultrasensitive on-site quantification of SAMs. For example, one method used chemiluminescent biosensors integrating a competitive lateral flow immunoassay and a charge-coupled device camera to detect FBs [264,265]. Another method uses gold nanoparticles or quantum dots nanobeads based on monoclonal antibodies against fumonisin and allows rapid detection of this mycotoxin in one step [266-268]. Furthermore, a loop-mediated isothermal amplification (LAMP) assay, based on the detection of *fum10*, could specifically detect the genes involved in FB~2~ biosynthesis in *Aspergillus* species that could be evaluated using the naked eye in a short time [269]. This method was also applied to detect FB~1~ targeting the *FUM1* gene in *Fusarium* [270]. The direct detection of genes involved in the biosynthesis of SAMs in agriculture production systems allows broad evaluations of the potential fumonisin-producing strains in food and feed products. To this end, multiplex PCR has shown great promise for detection of multiple fumonisin-producing *Fusarium* and *Aspergillus* species [271-273].

While the current focuses are on the known SAMs with known toxicities, there is increasing evidence that some of these toxins are masked and not easily detected or quantified. To ensure food safety, both the free forms and the masked forms of mycotoxins should be detected and quantified. The masked mycotoxins are usually modified forms of the mycotoxins by plant enzymes during infection and are not typically detectable during routine analysis. For example, the masked mycotoxins may conjugate with polar substances, store in the vacuole in the soluble form, or bind to macromolecules, and thus change their physiological properties. While the masked mycotoxins are often less toxic than the unmasked forms, they could be easily converted to the unmasked toxin forms, including during food digestion [274,275]. The most representative masked mycotoxins are the modified forms of Zearalenone (ZEN), DON, and fumonisins [276-279]. The so-called hidden fumonisins could form non-covalent bonds with food macro-constituents such as those in starch-based products. In certain situations, the masked fumonisins may be present in food at quantities much higher than the free forms. Many factors could influence the relative portions of the SAMs in masked forms, including crop growth conditions and food storage and processing techniques [279-282]. These hidden dangers require that novel method(s) be developed to allow the detection and quantification of the masked forms of SAMs as well as other mycotoxins.

## 11. Concluding Remarks

SAMs are highly toxic fungal compounds that have attracted significant attention from broad communities. They have toxicities to both plants and animals. These SAMs-producing pathogens are widely distributed in nature and closely related to agricultural production. Since its discovery in the mid-1980s, fumonisin has been among the mycotoxins with the greatest concern. As of now, Scopus database citations of fumonisin are above 5000, including 500+ reviews. SAMs are a series of compounds with structural similarity to sphingosine. As detection methods improve, additional new analogs have been continuously discovered. Research so far has shown that the toxicity and activity of SAMs are dependent on their structures. In this review, we summarized the detoxification method
based on their structural properties using chemical, biological, and physical strategies. The toxicity of SAMs is mostly due to their inhibitory effects on CerS, disruption on sphingolipid metabolism, and initiation on PCD. Except for the adverse effect of SAMs on animals and humans, its phytotoxicity (e.g., AAL-toxin) could potentially be used for herbicide development and a model for studying the molecular mechanism of PCD in plants. Horizontal gene transfers on the SAMs biosynthesis gene cluster seemed widespread in these toxin-producing fungi, especially among *Fusarium* spp. Such phylogenetic distribution patterns suggest that there are potentially other fungi capable of producing SAMs, including their various modified forms. These and other issues require continued efforts from the scientific community on SAMs.

**Author Contributions:** J.X. conceived the review and finalized the manuscript, J.C. conducted the literature search and drafted the manuscript, Z.L. drafted all the figures and tables, Y.C. coordinated the format and submitted the manuscript to the journal, C.G., L.G., and T.W. helped to revise the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Key R&D Program of China (N0: 2018YFD0201106), Natural Science Foundation of Hunan (2020JJ5637) and Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (Grant CAAS-ASTIP-2015-IBFC).

**Conflicts of Interest:** The authors declare no conflict of interest.

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