Endophytic fungi: a reservoir of antibacterials

Multidrug drug resistant bacteria are becoming increasingly problematic particularly in the under developed countries of the world. The most important microorganisms that have seen a geometric rise in numbers are Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus faecium, Penicillin resistant Streptococcus pneumonia and multiple drug resistant tubercule bacteria to name a just few. New drug scaffolds are essential to tackle this every increasing problem. These scaffolds can be sourced from nature itself. Endophytic fungi are an important reservoir of therapeutically active compounds. This review attempts to present some data relevant to the problem. New, very specific and effective antibiotics are needed but also at an affordable price! A Herculean task for researchers all over the world! In the Asian subcontinent indigenous therapeutics that has been practiced over the centuries such as Ayurveda have been effective as “handed down data” in family generations. May need a second, third and more “in-depth investigations?”


INTRODUCTION
The last two decades have witnessed a rise in the numbers of Methicillin resistant Staphylococcus aureus (MRSA), Vancomycin resistant Enterococcus faecium (VRE) and Penicillin resistant Streptococcus pneumoniae (PRSP) and a variety of antibiotics (Menichetti, 2005). New drugs such as Linezolid and Daptomycin have already acquired resistance (Mutnick et al., 2003;Skiest, 2006). MDR-and XDR-TB (Gillespie, 2002;LoBue, 2009) are emerging global threats, being difficult to diagnose, expensive to treat and with variable results. Rice (2008) reported that the ESKAPE organism's E. faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, P. aeruginosa, and Enterobacter species are the main causative agents of infections in a majority of US hospitals. To combat all these continuing developments, a search for new and novel drugs scaffolds remains the high priority activity.
Eighty five years after the discovery of Penicillin in 1929, scientists all over the world continue to investigate natural products. The novelty of structures and scaffolds, their varied bioactivities plus their abilities to act as lead molecules is immense. According to Newman and Cragg (2012), in the years 1981-2010, ∼50% of all small molecules originated from natural products. Mainly antibacterial, anticancer, antiviral and antifungals compounds from natural sources such as plant, fungi and bacteria themselves. The extraordinary advantages of natural products as sources of biotherapeutics is beyond question.
Though diverse chemical compounds with equally diverse scaffolds and bioactivities have been reported from fungi over the years, the vast group still remains to be fully exploited. Out of ∼1 million different fungal species only ∼100,000 have been described (Hawksworth and Rossman, 1997). Dreyfuss and Chapela (1994) estimated that endophytic fungi, alone could be ∼1 million. The genetic diversity of fungal endophytes may be a major factor in the discovery of novel bioactive compounds (Gunatilaka, 2006). The true potential of these endophytes is yet to be trapped.
From the first reports of isolation from the Lolium temulentum typically known as Darnel (ryegrass) by Freeman (1904), to the latest one from Antarctic moss (Melo et al., 2014), endophytic fungi have attracted the attention of botanists, chemists, ecologists, mycologists, plant pathologists and pharmacologists. It is estimated that each and every of the almost 300,000 plants that exist, hosts one or more endophyte (Strobel and Daisy, 2003). They occur everywhere, from the Arctic to Antarctic and temperate to the tropical climates. Endophytes reside in internal tissues of living plants but this association does not cause any immediate, overt, negative effects on the host plant (Bacon and White, 2000). According to Aly et al. (2011), the endophyte-plant host relationship is a balanced symbiotic continuum, ranging from mutualism through commensalism to parasitism. Many endophytic fungi remain quiescent within their hosts until it stressed or begins to undergo senescence. At this juncture the fungi may turn pathogenic (Rodriguez and Redman, 2008).
The need for novel antibacterials to combat this increasing variety of infections becomes a priority endeavor. Endophytic fungi may be an important source for such biotherapeutics like new antibacterials against Mycobacterium tuberculosis especially in poverty ridden tropical countries of Asia. Here the need could also involve a nutritional efforts to boost the immunity in the population. Many of the compounds with their host plants are shown in Table 1.

ANTIBACTERIALS FROM ENDOPHYTIC FUNGI COMPOUNDS FROM ASCOMYCETES
Ascomycetes are an important class of fungi where there is formation of ascospores. Some genera of this class are prolific producer of bioactive metabolites. The genus Pestalotiopsis exists as an endophyte in most of the world's rainforests and is extremely biochemically diverse. Some examples of products from this group are Ambuic acid (1) and its derivative (2) (Figure 1) isolated from a Pestalotiopsis sp. of the lichen Clavaroid sp. Compounds (1) and (2) are active against S. aureus (ATCC 6538) with IC 50 values of 43.9 and 27.8 μM, respectively (the positive control Ampicillin showed an IC 50 value of 1.40 μM) (Ding et al., 2009). Pestalotiopen A (3) (Figure 1), from Pestalotiopsis sp. of the Chinese mangrove Rhizophora mucronata exhibited moderate antimicrobial activity against Enterococcus faecalis with an MIC value between 125 and 250 μg/mL (Hemberger et al., 2013).
Phomopsis, another important genus exists as an endophyte in most plants and is also extremely biochemically diverse. Examples of bioactive metabolites from this endophyte are Dicerandrol A (6), B (7), and C (8) (Figure 1) from Phomopsis longicolla of the mint Dicerandra frutescens. They exhibit zones of inhibition of 11, 9.5, and 8.0 mm against B. subtilis respectively and 10.8, 9.5, and 7.0 mm respectively against S. aureus when tested at 300 μg/disc (Wagenaar and Clardy, 2001).
Phomoxanthones A (12) and B (13) (Figure 1) were obtained from Phomopsis sp. BCC 1323, of the leaf of Tectona grandis L., from the Mee Rim district of Chaingmai Province, Northern Thailand. These compounds show significant "in vitro" antitubercular activities with MICs of 0.5 and 6.25 μg/mL respectively against Mycobacterium tuberculosis H37Ra strain, in comparison to isoniazide and kanamycin sulfate (MICs of 0.050 and 2.5 μg/mL, respectively) that are used in clinics today (Isaka et al., 2001).
Phomoxanthone A (12) (Figure 1), was also isolated from a Phomopsis sp. of the stem of Costus sp. growing in the rain forest of Costa Rica. It has activity against Bacillus megaterium at a concentration of 10 mg/mL (radius of zone of inhibition of 3-4 cm) (Elsaesser et al., 2005).
Phomosines A-C (16-18) (Figure 2), three new biaryl ethers were obtained from Phomopsis sp. of the leaves of Teucrium scorodonia. All three compounds were moderately active against B. megaterium and E. coli in vitro, using 6 mm filter paper disc with 50 μl each of a 15 mg/mL solution (Krohn et al., 1995). The same compounds were obtained from Phomosis sp. of Ligustrum vulgare and showed activity against B. megaterium in vitro with 10, 10, and 7 mm zone of inhibition using 6 mm filter paper disc and 50 μg of compound (50 μL of 1 mg/mL) respectively .
3-Nitropropionic acid (35) (Figure 2) was isolated from several strains of endophytic fungus of the genus Phomopsis sp. obtained from six species of Thai medicinal plants ( Table 1) from the forest areas of Chiangmai, Nakhonrachasima, and Pitsanulok Provinces of Thailand. 3-Nitropropionic acid exhibits potent activity against Mycobacterium tuberculosis H37Ra with the MIC of 3.3 μM, but no in vitro cytotoxicity was observed toward a number of cell lines (Chomcheon et al., 2005). 3-Nitropropionic acid is known to inhibit isocitrate lyase (ICL), an enzyme required for fatty acid catabolism and virulence in M. tuberculosis (Muñoz-Elías and McKinney, 2005).
Phoma is another genus which produces diverse compounds. Here are some examples of bioactive compounds produced by this genus. Phomol (36) (Figure 3), a novel antibiotic, was isolated from a Phomopsis sp. of the medicinal plant Erythrina crista-galli. Phomol is active against Arthrobacter citreus and Corynebacterium insidiosum with MICs of 20 and 10 μg/mL respectively (Weber et al., 2004).
Phomodione (37), an usnic acid derivative was isolated from a Phoma sp. of Saurauia scaberrinae. Phomodione was found to be effective against S. aureus at a MIC of 1.6 μg/mL (Hoffman et al., 2008).
Dinemasones A(91) and B (92) (Figure 5), were isolated from Dinemasporium strigosum obtained from the roots of the herbaceous plant Calystegia sepium growing on the shores of the Baltic Sea, Wustrow, Germany. The above compounds showed antibacterial activities against B. megaterium .
Chlorogenic acid (142) (Figure 10) was isolated from the endophyte strain B5 a Sordariomycete sp. of Eucommia ulmoides. Eucommia ulmoides is a medicinal plant of China and one of the main sources of Chlorogenic acid. It has antibacterial, antifungal, antioxidant and antitumor activities .
Two new dihydroisocoumarin derivatives Aspergillumarins A (156) and B (157) (Figure 11) are produced by a marine-derived Aspergillus sp., of the mangrove Bruguiera gymnorrhiza collected from the South China Sea. Both show weak antibacterial activities against S. aureus and B. subtilis at 50 μg/mL .
Sanguinarine (196) (Figure 14), a benzophenanthridine alkaloid was obtained from the endophyte Fusarium proliferatum (strain BLH51) present on the leaves of Macleaya cordata of the Dabie Mountain, China. It has antibacterial, anthelmintic, and anti-inflammatory activities (Wang et al., 2014). It has antibacterial activities against the range of bacteria with MICs of 3.12-6.25 μg/mL against 15 clinical isolates of S. aureus while the MICs against of the two reference strains are 3.12 μg/mL for ATCC 25923 and 1.56 μg/mL for ATCC 33591.
The clinical isolates strains showed MIC values ranging from 31.25 to 250 μg/mL for ampicillin and 125-1000 μg/mL for ciprofloxacin. The treatment of the cells with sanguinarine induced the release of membrane-bound cell wall autolytic enzymes, which eventually resulted in lysis of the cell. Transmission electron microscopy (TEM) of MRSA treated with Sanguinarine show alterations in septa formation. The predisposition of lysis and altered morphology seen by TEM indicates that sanguinarine acts on the cytoplasmic membrane (Obiang-Obounou et al., 2011). The compound also has activity against plaque bacteria with MICs of 1-32 μg/mL for most species tested. The Electron microscopic studies of bacteria exposed to sanguinarine show that they aggregate and become morphologically irregular (Godowski, 1989).
Guanacastepene A (243) (Figure 18), a novel diterpenoid produced the fungus CR115 isolated from the branch of Daphnopsis americana growing in Guanacaste, Costa Rica, may prove to belong to potentially new class of antibacterial agents with activities against MRSA and VRE . Guanacastepene I (244) (Figure 18), was isolated from the same fungus is active against S. aureus (Brady et al., 2001).
Mirandamycin (265) (Figure 19) was obtained from isolate 1223-D, an unclassified fungus of twig of Neomirandea angularis of family Asteraceae. It is active against E.coli 25922, P. aeruginosa 27853, K. pneumoniae carbapenemase positive BAA-1705, MRSA BAA-976 and V. cholerae PW357 with MICs of 80, 80, >80, 10, and 40 μg/mL respectively (Ymele-Leki et al., 2012). Strobel et al. (2001) reported at least 28 volatile organic compounds (VOC) from the xylariaceaous endophyte Muscodor albus (isolate 620), of Cinnamomum zeylanicum from Lancetilla Botanical Garden near La Ceiba, Honduras. These VOC's are mixtures of gasses of five class's viz. alcohols, acids, esters, ketones and lipids. The most effective were the esters, of which, 1butanol, 3-methyl-acetate has the highest activity. The VOC's inhibited and killed certain bacteria, within a period of 1-3 days. Most test organisms were completely inhibited, and in fact killed. These includes Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis along with some fungal species. Strain of Muscodor namely Muscodor crispans of Ananas ananassoides (wild pineapple) growing in the Bolivian Amazon Basin produces VOC's; namely propanoic acid, 2-methyl-; 1butanol, 3-methyl-; 1-butanol, 3-methyl-, acetate; propanoic acid, 2-methyl-, 2-methylbutyl ester; and ethanol. The VOC's of this fungus are effective against Xanthomonas axonopodis pv. citri a citrus pathogens. The VOC's of M. crispans kill several human pathogens, including Yersinia pestis, Mycobacterium tuberculosis and Staphylococcus aureus. Muscodor crispans is only effective against the vegetative cells of Bacillus anthracis, but not against the spores. Artificial mixtures of the fungal VOC's were both inhibitory and lethal to a number of human and plant pathogens, including three drug-resistant strains of Mycobacterium tuberculosis (Mitchell et al., 2010). The mechanism of action of the VOC's of Muscodor spp. on target bacteria is unknown. A microarray study of the transcriptional response analysis of B. subtilis cells exposed to M. albus VOC's show that the expression of genes involved in DNA repair and replication increased, suggesting that VOC's induce some type of DNA damage in cells, possibly through the effect of one of the naphthalene derivatives (Mitchell et al., 2010).

Outlook
A definite, urgent and worldwide effort is needed to tackle the problems of the populations in third world and developing countries. MRSA, VRE, PRSP, ESCAPE organisms have spread through these countries over the years particularly due to immunocompromised populations. Mycobacterium tuberculosis is a major threat! and New and Novel drugs are a must!! Endophytic fungi may be an excellent source of such compounds. These organisms have a vast repertoire of diverse chemicals such as steroids, xanthones, phenols, isocoumarins, perylene derivatives, quinones, furandiones, terpenoids, depsipeptides and cytochalasins (Tan and Zou, 2001;Gunatilaka, 2006;Zhang et al., 2006;Guo et al., 2008).
A major challenge in Drug Discovery Program based on endophytic fungi lies in developing effective strategies to isolating bioactive strains. Strobel and Daisy (2003) suggested that areas of high biodiversity of endemic plant species may hold the greatest potential for endophytes with novel chemical entities. Tropical forests are some of the most bio diverse ecosystems and their leaves are "biodiversity hotspots" (Arnold and Lutzoni, 2007). The selection of plants is crucial. Those with medicinal properties should be given preference. Metabolites produced by fungi need to correlated with the plant genomics, thus allowing far better knowledge of biosynthetic pathways. This will also justify the production of metabolites rather than unproven hypotheses.
Identification of endophytic fungi using molecular analyses provides an opportunity to look for broad patterns in bioactivity not only at the genotype or strain level, but at higher taxonomic levels that may in turn assist in focusing on the association of metabolite with the plant.
The endophytic flora of the Indian subcontinent has been explored for their diversity but not enough for their bioactive metabolites. The published work is scanty (Puri et al., 2005;Deshmukh et al., 2009;Khrawar et al., 2009;Periyasamy et al., 2014). There is a need for groups from different scientific discipline (mycologist, chemist, toxicologist, and pharmacologist) to engage in this search process. Enormous natural wealth exists in the world's tropical forests, but disparity exists between developed countries with their financial resources and biodiversity rich countries with underdeveloped economy and limited funds. May be funding agencies need to look at such aspects.
The need of a more and larger collection of fungal endopytes is suggested. Bioactive metabolite metabolites from such collections could yield leads for pharmaceutical and agricultural application.
What emerges is the essential bonding of various discipline of biology and chemistry into cohesive target delivery vehicles.