Bioactive Novel Natural Products from Marine Sponge: Associated Fungi

Abstract

Marine sponges are distributed in the water, from the intertidal zones to thousands of meters deep. They are primitive multicellular invertebrates that live in benthic environments and are bound to solid substrates. Filter feeders, sponges have many microscopic pores on their surface, which allow water to enter and circulate via a network of canals where microbes and organic particles are filtered out and absorbed. Marine fungi are widespread in the oceans and colonize different ecological niches; they are found associated with organisms of all trophic levels and can act as saprobes, symbionts, and parasites. Compared with other marine microorganisms, marine fungus is relatively understudied. Fungi associated with sponges have been discovered to be a promising source of pharmacologically active compounds with unique anticancer, antibacterial, and antiviral properties.

1. Introduction

The ocean is a unique resource that provides a wide range of natural products. The greatest biodiversity is found in ecosystems with high species diversity and population density, such as rocky coasts, kelp beds, and coral reefs [1]. Marine sponges are benthic animals that live in a variety of marine environments. Sponge species diversity is significantly higher in tropical coral reef environments. The ocean is called the “mother of origin of life,” and an enormous proportion of all life on Earth exists within the oceans [2].

Marine sponges (Porifera) are the earliest living animal phylum and represent the very beginning of metazoan development. Sponge feeding, as a sedentary organism, sequesters food particles. They can pump and filter a large volume of seawater through a unique and highly vascularized canal system (G [3]).

Sponges are divided into three groups: the Calcarea (five orders and 24 families), Demospongiae (15 orders and 92 families), and Hexactinellida (five orders and 92 families) (six orders and 20 families). About 15,000 sponge species have been identified so far, but their total diversity can be much larger. The 95% of them live in the ocean, with only around 1% existing in freshwater [4].

Sponges are a great place to live not only for macro-organisms such as worms, shrimp, and crabs, but also for a variety of microorganisms, which live in the canals, between cells, and even inside the cells. Large numbers of microorganisms, such as bacteria, algae, phytoplankton, and fungi, become key components of sponges’ natural diets during the filter feeding process. In addition, sponges also have diversified microbiome that accounts for up to 40% of the sponge biomass. Sponge-microbe symbioses are considered to promote sponges by providing sustenance, transporting waste products or active metabolites, chemical defense and contribute to mechanical structure (in general) [5].

Microorganisms found in marine animals have a huge potential as a source of bioactive compounds [6]. Sponge relationships are important for exploring biologically active substances that can be used to develop pharmaceuticals, agrochemicals, and biochemical reagents, as well as their lead molecules. It is hypothesized that symbiotic marine microorganisms harbored by sponges are the original producers of these bioactive compounds [7].

Marine fungi belong to a diversity of families; however, they appear to be in low quantities in seawater (in relative to bacteria) and contribute for only 0.6% of the global fungal diversity. The definition of a marine fungus is broad and based on the habitat [8]. Obligate marine fungi are those that grow and sporulate exclusively in a marine or estuary ecosystem; facultative marine fungi are those that can grow and sporulate including both freshwater and terrestrial ecosystems.

The potential fungal origin of a mitochondrial intron presents in the sponge Tetilla sp., which was thought to have emerged from a cross-kingdom horizontal gene transfer, has also been viewed as indirect evidence for a symbiotic relationship between fungus and a sponge [7, 9]. Indirect evidence of interactions between marine sponges and fungi was also provided by the detection of fungal introns in the genomes of some marine sponge species that were most probably acquired by horizontal gene transfer [9].

Marine sponges provide another habitat for fungi [5, 10], knowledge of sponge-associated fungal diversity remains scarce [11]. Marine fungi have provided a major source of new biological natural products, because of their characteristic properties with reference to temperature, nutrients, competition, and salinity [12]. Fungi have been repeatedly isolated from many sponge species [13, 14]. An extensive survey also revealed that there are thousands of other fungi-derived bioactive metabolite families that are yet to be known [15]. Sponge-associated fungi have been reported to create structurally distinct bioactive compounds compared with terrestrial [16, 17].

Considering the fact that researchers do not know much about the fungal life cycle in sponges and other environmental fungi (Richards et al., 2012), it is fascinating to hypothesis about the role of sponge-associated fungi. Many sponge-derived fungi have been found to produce bioactive substances, indicating that they may be involved in chemical host protection [18].

Thirunavukkarasu et al. [19] investigated the filamentous fungal symbionts of 10 marine sponge species from Rameswaram, southern India. The findings indicate that fungal symbionts of marine sponges are extremely diverse. Acremonium, Alternaria, Aspergillus, Cladosporium, Fusarium, and Penicillium were frequently isolated. A few fungi produced acetylcholinesterase inhibitors. Fungi associated with marine sponges have been investigated more avidly for their potential technological applications owing to their ability to synthesize metabolites of novel molecular architectures and bioactivities [20, 21, 22, 23].

Several Antarctic sponges of the phylum Ascomycota were a rich source of associate fungi and novel bioactive compounds, with some of them having antibacterial, antitumoral, and antioxidant potential, according to a study on the fungal community using a culture-dependent technique [24].

Many findings proved that sponge-derived fungi are the true biosynthetic origin, able to produce secondary metabolites such as jasplakinolide [25]. Number of fungal strains from marine sponges have been isolated and belong to three phyla, namely Ascomycota, Zygomycota, and mitosporic fungi [20, 26, 27].

1.1 Cultivation of sponge-associated fungi

Sponge tissues are sensitive, and using harsh surface sterilization techniques to isolate their endosymbionts causes sponge disintegration resulting in symbiont death [19]. Most of the surface contaminants can be removed with either a milder sterilization using 70% ethanol for 30 seconds and then washing with seawater or a thorough washing of sponge tissue segments in sterile seawater [28]. Fast-growing fungi, such as Aspergillus or Penicillium, grow in these conditions, whereas slow-growing species, if present, may go undetected. Weeding out fast-growing fungi or improving the isolation medium with Rose Bengal, with an antibiotic as in isolation techniques for endophytic fungi [10].

In traditional plating method, one gram of sponge sample was mixed in 9 and 99-ml sterile water blank, respectively. This suspension was serially diluted up to 10⁻⁴.^. Diluted sample was taken from 10⁻³ and 10⁻⁴ dilutions and was pour plated with 15 ml–20 ml potato dextrose agar (PDA) and incubated at room temperature (28 ± 2°C) for 5 days [29, 30]. Instead, to isolate fungal symbionts, the sponge’s water can be squeezed onto nutritive agar medium and cultured [21]. Plate out comminuted sponge tissues on nutritive agar medium as another method of isolating intracellular fungus (J. F. [31]).

Wei et al. [32] applied cultivation-dependent approach to study the fungal diversity in the Hawaiian sponges Gelliodes fibrosa, Haliclona caerulea, and Mycale armata dependent approach. The cultivated fungal isolates belonged to at least 25 genera of Ascomycota and one of Basidiomycota, representing eight taxonomic orders. Cultivated fungal isolates were divided into three groups: sponge-generalists (found in all sponge species), sponge-associates (found in more than one sponge species), and sponge-specialists (found only in one sponge species) [33]. Caballero-George et al. [34] isolated a total of 369 marine sponges that were collected along Panama’s coasts in high biodiversity areas. A total of 2263 fungal isolates were recovered from the sponges. Calabon et al. [35] found that Aspergillus was the most dominant genus among 22 genera of ascomycetes isolated from mangrove-originated sponges in Aklan, Philippines, with 23 isolates, followed by Mycelia (n = 21 isolates) and Penicillium (n = 14 isolates).

Marine fungi are especially adept at living on or inside other living organisms such as sponges, corals algae, and even other fungi [36]. Unique metabolic pathways have evolved in halotolerant marine fungal species that are responsive to salt concentrations. Fungi must have osmoregulatory mechanisms that signal the synthesis of polyols and amino compounds while also increasing the concentration of cytoplasmic ions in order to develop in the marine environment. Because the biosynthesis of these osmoregulating solutes is energy-intensive, fungus may release least secondary metabolites or produce them at a slower rate while exposed to high salinity levels [37].

The Endolithic fungus genus Koralionastes is always found in close association with encrusting sponges, which is an interesting observation. Future research on the relationships between fungi and their marine hosts will help our understanding of the marine ecosystem, and it may lead to improved collecting methods and the isolation of chemically unknown species [20].

1.2 Morphological and molecular identification of fungi

Sponge-associated fungi, in particular, have been proven to be the richest source of various bioactive metabolites and novel metabolites. The ecological function and connections of sponge fungi, on the other hand, are mostly unknown. More specific evidence for sponge-associated fungal functions is required. Fungal functions linked with sponges must be provided. The sponge-associated fungal function analysis can employ an activity-based analysis technique, but it will be limited in some cases because the culture condition during the bioassay may not be optimal for the production of linked bioactives.

For natural product exploration, many sponge marine-derived fungi have been isolated by many researchers. It was identified primarily by microscopic observation using wet mount and lacto phenol cotton blue stain preparations. Traditional monographs, polyphasic taxonomic approach, and molecular nucleotide sequences of marker DNA such as ITS, 18 s, and others are used to identify marine fungus strains [24, 38, 39]. Though, ITS rDNA regions are most often used to identify species and strain-level fungal diversity. DNA barcode data for approximately 100,000 fungal isolates were generated using sequences of two nuclear ribosomal genetic markers, the Internal Transcribed Spacer and 5.8S gene (ITS) and the D1/D2 domain of the 26S Large Subunit (LSU).

1.3 Taxonomic databases resources for marine fungi

The internet has become a vital source of information for millions of individuals. Over the last few decades, fungal research has broadened its scope, generating a wealth of information that has led to the establishment of many site dedicated to various aspects of mycology and also exclusively marine fungi such as http://www.marinespecies.org/.and,http://pubs.rsc.org/marinlit.

All genera of fungi, including marine fungi, are classified and details are provided in the database (http://fungalgenera.org/). The Indian marine fungal database (Figure 1) is another resource (www.fungifromindia.com/), which is linked to MycoBank and provides 233 strains of marine fungi identified in India. The World Register of Marine Species (WoRMS) (www.marinespecies.org) aims to provide a comprehensive and definitive listing of all marine life forms’ names [40, 41, 42]. www.marinefungi.org is a marine web portal. This web portal provides researchers with access to the classification, detailed descriptions, and worldwide distribution of all known marine and marine-derived fungi [43].

Figure 1.

Indian marine fungi database (www.fungifromindia.com).

2. Bioactive natural metabolites from sponge-associated fungi

Marine sponge-associated fungi are one such group that has been reported to be a crucial and invaluable source of novel therapeutic agents possessing several bioactive properties including free radical scavenging activity, neuritogenic activity, anticancer activity, and kinase inhibition, etc. The exploration of fungal metabolites has significantly increased after marine fungi, especially sponge-associated fungi have been reported to produce structurally unique bioactive compounds [44, 45].

The first metabolite reported from a sponge-derived fungus was Trichoharzin, which was isolated from a strain of Trichoderma harzianum associated with the sponge Mycale cecilia in 1993.

2.1 Anticancer compounds

The diversity of biochemical properties of sponges had been demonstrated by the continued discovery of novel compounds, having pharmacological properties [46]. The marine-derived fungus Aspergillus sp., which was obtained from the sponge Xestospongia testudinaria, was collected from the South China Sea that gave two phenolic bisabolane sesquiterpenoid dimers, disydonols A and C exhibited in vitro moderate cytotoxicity toward HepG- 2 and Caski human tumor cell lines (IC50 values of 9.31 and 12.40 μg/mL) [47]. Fungi Stachylidium spp. was isolated from the sponge Callyspongia cf. C. flammea. Chemical investigation of the bioactive fungal extract led to the isolation of the novel phthalimidine derivatives marilines A1 and A2. Both enantiomers, marilines A1 and A2 inhibited human leukocyte elastase (HLE) with an IC50 value of 0.86 μM [48]. The fungal species Aspergillus, which is widespread across the globe, is also the major source of bioactive molecules in marine sponges. The bulk of the 680 fungal strains derived from 16 sponge species around the world are mostly from the genera Aspergillus and Penicillium [49].

Three marine sponges, Tedania anhelans, Myxilla arenaria, Callyspongia fibrosa, were collected from Vizhinjam and Kovalam in Kerala. Aspergillus sp. MCCF 103, Aspergillus sp. MCCF 111, Aspergillus sp. MCCF 114, Penicillium sp. MCCF 115, and Aspergillus sp. MCCF were isolated and identified. These strains have significant cytotoxic activity on NCI-H460 lung cancer cells lines [50]. Yellow-colored compounds 2-(2′, 3-epoxy-1′,3′-heptadienyl)-6-hydroxy-5-(3- methyl-2-butenyl) benzaldehyde and 1,8-dihydroxy-6-methoxy- 3-methyl-9,10-anthracenedione (physcion) are extracted from the marine sponge Mycale sp., associated fungus Eurotium cristatum [48].

Violaceimides A and B, two methyl succinimide-based sulfur-bearing compounds, were isolated from the sponge-associated fungal strain Aspergillus violaceus WZXY-m64-17.

Both compounds suppressed human leukemia U937 growth with IC50 values of 5.3 ± 0.4 and 1.8 ± 0.6 mM, respectively, as well as human colorectal cancer cell HCT-8 with IC50 values of 1.5 ± 0.28 mM [51]. Mactanamide, a diketopiperazine alkaloid, was isolated from the marine sponge Stylissa sp. derived fungus Aspergillus flocculosus, which was collected in Vietnam. The isolated compound was screened for antiproliferation activity, and it proved a significant effect of non-cytotoxic suppression on osteoclast differentiation (Shin et al., 2017).

Preussin, a hydroxyl pyrrolidine derivative, was isolated from Aspergillus candidus KUFA 0062, a fungus associated to sea sponges. The antiproliferative and cytotoxic activities of this pyrrolidine derivative have been tested in breast cancer cells (SKBR3, MCF7, and MDA-MB-231), as well as MCF12A, a non-tumor cell line. Various assays have been used to examine cell morphology for ki67 and caspase-3, as well as 3D (multicellular aggregates) and 2D (monolayer) culturing tests. Preussin-exposed cells morphological study indicated apoptosis, which was confirmed by caspase-3 immunohistochemistry. 3D culture cells were less sensitive, and preussin-exposed cells morphological analysis revealed apoptosis, which was confirmed by caspase-3 immunohistochemistry [52].

Bioactive component methyl averantin is produced by Aspergillus versicolor in association with the sponge Petrosia sp. This secondary metabolite belongs to the anthraquinone family. Methyl averantin has a high cytotoxic activity, with an IC50 .4–1.1 μg/ml in cancer cell lines such as A-549, HCT-15, SK-MEL-2, SK-OV-3, and XF- 498 [53]. The compounds heterocornols AC, FH, methyl(2formyl3hydroxyphenyl) propanoate, agropyrenol, and vaccinol G have been isolated from the fungus Pestalotiopsis hetero cornis XWS03F09 associated with the marine sponge Phakellia fusca and have cytotoxicity against four human cancer cell lines and antimicrobial activity [54]. The Asteltoxins E and F polyketides were isolated from the marine sponge-derived fungus Aspergillus sp. SCSIO XWS02F40. With IC50 values of 6.2 ± 0.08 and 8.9 ± 0.3 mM, respectively, asteltoxin E and F demonstrated potent antiviral activity against influenza virus A subtype H3N2 (A/H3N2). Furthermore, asteltoxin E reported to inhibit the activity of influenza virus A subtype H1N1 (A/) [55].

The fungus Arthrinium arundinis ZSDS1-F3 was isolated from the marine sponge Phakellia fusca in the Xisha Islands of China, from that cytochalasin K was extracted that showed cytotoxicity against K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, HeLa, and MOLT-4 cell lines, with IC50 values of 10.5, 13.7, 10.9, 19.1, 11.1, 47.4, and 11.8 μM respectively [56].

Elissawy et al. (2017) extracted Curvularin, Cyclo(L- Pro-L-Ile), and Cyclo(L-Tyr-L-Pro), from the fungus Aspergillus versicolor isolated from the black sponge Spongia officinalis, which play inhibitory activity against HCV NS3/4A protease.

Therapeutic enzymes are used to treat diseases such as cancer, severe disorders such as autism, chronic lung disease, and multiple sclerosis, although cancer seems to be the most potential therapeutic application for enzymes. Therapeutic enzymes, it seems out, have a unique ability to facilitate high-affinity interactions with unrelated cancer-related proteins. Endophytic fungi recovered from the marine soft sponge Aplysina fistularis, produce L-asparaginase [57].

2.2 Antimicrobial compounds

Polyketide-derived alkaloids, terpenes, peptides, and combined biosynthetic chemicals are prominent classes of secondary metabolites produced by marine sponge-derived fungus. Miriam et al. [58] isolated several bioactive secondary metabolites from the fungi P. raistrikii associated Axinella cf. corrugate (sponge), including (4-methoxy-5-3-methoxybut-1-enyl)-6- methyl-2H- pyran-2-one, a new metabolite isolated from the Penicillium paxilli strain MaGK, Norlique xanthone, also known as 1, 3,6- trihydroxy-8-methyl-9H-xanthen-9 [32].

Triazolic compound was extracted from Aspergillus clavatus MFD15 that is associated with marine sponges. This compound is found to 50% inhibit E. coli, S. aureus, and S. epidermidis [59].

Fugal extract of Aspergillus sydowii from the waters of Riung, East Nusa Tenggara, Indonesia, was associated with marine sponge Axinella sp. and showed antibacterial activity. These extracts have bioactivity against E. coli and S. aureus. The maximum zone is obtained from MG KN-15-3-1-3 extract, with inhibition zones of 10.71 mm and 10.98 mm against E. coli and S. aureus [60]. Likewise, Austalide U, a meroterpenoid, has been produced by the sponge-derived fungus Aspergillus aureolatus HDN14-107. It showed antiviral efficiency against A/H1N1 virus [55].

Marine fungi are well known for producing a wide range of secondary metabolites, including numerous life-saving therapeutics [61]. MDR Escherichia coli has been linked to a variety of infectious diseases, as well as urinary tract infection, nosocomial bloodstream infection, meningitis, bacteremia, and gastrointestinal disease. There were 29 marine sponge-associated fungi isolated from nine sponges. Among 29 sponge-associated fungi screened, there were seven isolates that showed antibacterial activity against MDR E. coli [62]. Sponge-associated Aspergillus sp LS116 produced aspergill steroid A, a C23 steroid with a bicycloA/B ring (With an MIC value of 16 mg/mL1, this compound showed strong antibacterial activity against V. harveyi, indicating that aspergillsteroid A.) It could be considered one of the promising agents for aquatic disease control in the future Guo et al. [63].

Daldinia eschscholtzii is a fungus isolated from an Indonesian sponge called Xestospongia sp. located in Karimunjawa National Park in Central Java, Indonesia. Karimanone is a novel chromanone-type compound found and characterized from D. eschscholtzii, and it has three biosynthetically related metabolites. With an MIC of 62.5 g/ml for compound 2 and 125 g/ml for compounds 1, 3, and 4, all of the compounds were effective against a multidrug-resistant strain of Salmonella enterica ser. Typhi. [64].

Two cyclic tetrapeptides, sartory glabramides A and B and a bis-indoly lmethyl diketopiperazine, fell utanine A epoxide together with aszonalenin (3R)-3-(1H-indol-3-ylmethyl)-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione takakiamide), (11aR)-2,3-dihydro-1H-pyrrolo benzodiazepine- 5,11(10H,11aH)-dione and fellutanine A were isolated from the marine-derived fungus N. glabra KUFA 0702, which was isolated from the marine sponge Mycale sp., collected from the coral reef at Samaesarn Island, Thailand. The antibacterial activity of all identified compounds was tested against two bacterial pathogens, Staphylococcus aureus ATCC 46645 and E. coli ATCC 25922, as well as three fungal isolates, Aspergillus fumigatus ATCC 46645, Trichophyton rubrum ATCC FF5, and Candida albicans ATCC 10231 [65].

Chu et al. [66] isolated and identified the Aspergillus versicolor strain TS08 associated with South China Sea sponge Holoxea sp. also extracted the cyclo (L-Trp-L-Phe). The highest yield of cyclo (L-Trp-L-Phe), 13.24 mg/g (per crude extract of EtOAc), 2.51% of cell dry weigh, was obtained on the tenth day of the fungal cultivation. Scopel et al. [67] separated Arvoredol from Penicillium sp. F37, which was isolated from the marine sponge Axinella corrugate.

It is a chlorinated polyketide that contains 6,7-dihydro-4(5H) benzofuranone. Arvoredol inhibited biofilm formation by the human pathogen S. epidermidis by 40% at a concentration of 125 μgmL−1 by 40%.

The extract of Penicillium chrysogenum, obtained from the marine sponge Tedania anhelans, showed antimycobacterial activity [68], another sponge-associated Penicillium sp. produced citrinin, which has antibacterial and cytotoxic properties [69, 70]. Sabdaningsih et al. [71] also isolated P. citrinum WK-P9, a sponge-associated fungus, having been used to produce citrinin derivatives. Penicitrinol J. It was characterized, revealing a monomer connection previously unknown in citrinin derivatives. It inhibits the growth of B. subtilis JH642, B. megaterium DSM32, and M. smegmatis ATCC607.

On the one side, the extensive use of antibiotics has increased the prevalence of antibiotic resistance; on the other side, the use of immunosuppressive agents after transplantation has significantly increased the incidence of fungal infections. Phoma sp., a sponge-derived fungus, provides unique lactone compound capable of inhibiting several human pathogens such as C. albicans and Aspergillus fumigatus [18]. Curvularia lunata, a fungus, was isolated from the sponge Cinachyrella australiensis from the Karimunjawa Islands in Indonesia. C. lunata fungal extract demonstrated promising antibacterial activity against MDR S. pneumoniae [72].

Cytotoxic polyketides compounds were extracted from the sponge-derived fungus Aspergillus versicolor. Bioactivity-guided fractionation was used to isolate a new peptide in a subsequent investigation. Approximately 20 peptides have been reported from sponge-derived fungi, including efrapeptins Eα, H, RHM3, RHM4 (from two fungi, Acremonium sp. and Metarrhizium sp),4 homodestcardin (from Fusarium graminearum),5 clonostachysins A and B (from Clonostachys rogersoniana),6 a cyclodepsipeptide (from a Clonostachys sp),7 linear octapeptides (from an Acremonium sp),8 petrosifungins A and B (from Penicillium brevicompactum),9 fellutamides A and B (from Penicillium fellutanum),10 and halovirus (from a Scytalidium sp). Fungi Penicillium chrysogenum and Stachybotrys chartarum derived from sponge have been found to inhibit at different stages of the HIV viral cycle [73].

2.3 Antifungal activity

Peniciadametizine A and B were extracted from Penicillium adamatzioides, the marine sponge and have antifungal activity against Alternaria brassicae [13].Penicillium cf. montanense, a marine fungus that produces xestolactone B, is an associated fungus of the marine sponge Xetospongi aexigua. This compound is antifungal against C. albicans [74].

Sixty-seven sponges were collected from four different areas of Indonesian water. For screening the active isolates, an antagonistic test was performed against Malassezia furfur, Trichophyton sp., and C. albicans using the cross-streak method. Lampung Bay, Seribu Islands, Karimunjawa Islands, and Wakatobi Island had sponge-isolates ratios of 106%, 90% 210%, and 115%, respectively. The sponge collected from the Wakatobi Islands has one of the most active isolates against M. furfur, Trichophyton sp., and C. albicans [71].

A number of compounds synthesized by fungal symbionts from sponges may have agricultural implications. Penicillium adametzioides AS-53 produces peniciadametizine A, which is particularly active against the plantpathogenic fungus Alternaria brassicae (MIC 4.0 g/mL) [75]. Fungi isolated from sponges proved to be bioremediation agents, for instance by degrading the pesticide DDD (1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane), nowadays banned but still persistent in the environment [76].

3. Conclusion and future prospective

Marine environment represents an untapped source of fungal diversity, in comparison to sponge-associated prokaryotic microorganisms; there are few reports on the diversification of sponge-associated fungi. Marine sponge-associated fungi are rich in metabolites, which are less understood. Fungi associated with sponges are the most potent source of new natural compounds and display diverse biological activities. Based on recent research studies, marine fungal metabolites will find application toward pharmaceuticals, cosmeceuticals, nutraceuticals, etc.; from this review, it is also important to remember that secondary metabolite profiles differ from the same fungus species originating from various sponge species. As a conclusion, a systematic search for fungus and fungal metabolites in sponge species from various geographical regions is an essential step in bioprospecting.