Marine Bioactive Compounds against Aspergillus fumigatus: Challenges and Future Prospects

With the mortality rate of invasive aspergillosis caused by Aspergillus fumigatus reaching almost 100% among some groups of patients, and with the rapidly increasing resistance of A. fumigatus to available antifungal drugs, new antifungal agents have never been more desirable than now. Numerous bioactive compounds were isolated and characterized from marine resources. However, only a few exhibited a potent activity against A. fumigatus when compared to the multitude that did against some other pathogens. Here, we review the marine bioactive compounds that display a bioactivity against A. fumigatus. The challenges hampering the discovery of antifungal agents from this rich habitat are also critically analyzed. Further, we propose strategies that could speed up an efficient discovery and broaden the dimensions of screening in order to obtain promising in vivo antifungal agents with new modes of action.


Introduction
Aspergillus fumigatus is a saprophytic mold commonly found in the environment, with spores that are very light and easily disseminated [1]. It is also a potentially dangerous opportunistic pathogen that is reported as being the number one causative mold for mycoses, especially in immunocompromised patients [2]. Invasive aspergillosis (IA) caused by A. fumigatus is a severe systemic infection with high mortality and morbidity rates. IA has an annual global incidence of over 200,000, and the mortality rate could reach almost 100% among certain categories of patients if not properly treated [2][3][4].
With the already existing challenge of having only a limited repertoire of antifungal drugs for the treatment of aspergilloses, the continuous rise of drug resistance in A. fumigatus strains [5][6][7] further aggravates this challenge. Reports of A. fumigatus developing a resistance, in both clinical [8,9] and environmental [10,11] isolates, to antifungal drugs such as amphotericin B [12], azole-class drugs [13,14] and echinocandins [5,15], as well as developing a multidrug resistance [16], are increasing annually.
In the UK for example, Public Health England reported over a five-fold increase in A. fumigatus isolates that showed resistance to itraconazole (Minimum Inhibitory Concentration (MIC) ≥ 2.0 µg/mL) very weak activity against A. fumigatus, with MICs of >64 µg/mL [83]. On the other hand, extracts from some streptomycetes [86,87], and other bacteria like Micrococcus sp., Flavobacterium sp. and Streptomyces sp. [64], have also been demonstrated as possessing very effective activities against A. fumigatus (Table 2). More interestingly, some of these extracts also showed promising activities against A. fumigatus strains with multidrug resistance (MDR) ( Table 2).

A. fumigatus Effective Compounds from Marine Sponges
Marine sponges, such as Theonella swinhoei, Siliquariaspongia japonica and Microscleroderma herdmani, are other categories of interesting marine organisms demonstrated to produce effective antifungal compounds against A. fumigatus. Compounds such as Aurantosides E, A, B and Microsclerodermin B are the best antifungal bioactive compounds against A. fumigatus, with MICs of 0.04, 0.16, 0.16 and 0.6 µg/mL, respectively [77,88] (Table 3; Figure 2). Moreover, other Microsclerodermins (A, J and K) and Swinhoeiamide A also exhibit a good antifungal activity against A. fumigatus, with MICs ranging between 1 to 10 µg/mL [77,89] (Table 3).

A. fumigatus Effective Compounds from Marine Algae
Both compounds and extracts from several marine algae have been demonstrated to exhibit antifungal activities against A. fumigatus.

A. fumigatus Effective Compounds from Sea Cucumbers
Several triterpene glycosides ( Figure 4) with good antifungal activities against A. fumigatus have been isolated and characterized from sea cucumbers such as Holothuria scabra, Actinopyga lecanora, several species of Bohadschia, etc. [80,81,96,97]. Among the best bioactive compounds isolated from these marine echinoderms, potent activities with an MIC 80 ranging from 1.0 to 4.0 µg/mL have been recorded in in vitro screenings (Table 5). Triterpene glycosides therefore present very good prospects for future antifungal drug development.
Besides characterized compounds, both crude and partially purified extracts from different sea cucumbers have also been demonstrated to exhibit varying levels of antifungal activities against A. fumigatus. Ismail et al. [79] reported that crude and semipurified extracts from both aqueous body fluid extracts and methanolic wall extracts from Holothuria polii displayed varying activities against A. fumigatus in a concentration-dependent manner. Methanolic body-wall crude extracts showed better activities than those of aqueous body fluid. However, there was no difference in activities among the extracts after purification. Similarly, Adibpour et al. [98] also recorded an antifungal activity against A. fumigatus from Holothuria leucospilota body-wall and coelomic fluid extracts but none from its cuvierian organs' extracts.

A. fumigatus Effective Compounds from Marine Fungi
A few studies have reported bioactive compounds from marine fungi against pathogenic molds. With reference to A. fumigatus, to the best of our knowledge, only the marine fungus Phoma sp. produced a characterized bioactive compound, YM-202204, with an IC 80 of 12.5 µg/mL against A. fumigatus [69]. We propose that most fungi do indeed produce necessary antagonism metabolites against their fellow fungi if "they feel threatened" or if there is competition. That is why we have proposed several enhanced pathways (e.g., the coculture approach) for isolating and screening bioactive compounds (Figures 5 and 6).

Challenges and Future Prospects
One of the biggest challenges facing the discovery of effective antifungal agents against A. fumigatus and other pathogenic microorganisms from a rich marine environment is the limitation on culturable organisms. It has been proven that the vast majority of microorganisms in nature cannot be isolated through the usual cultural techniques and are therefore labelled as unculturable microorganisms [100,101], where marine microbes predominate. Therefore, obtaining these viable but unculturable marine microbes by using several methods, including molecular approaches [102] and simpler techniques such as a long incubation with low nutrition [101], will certainly open a new vista of opportunities for discovering new antimicrobial agents. This may consequently lead to obtaining and evaluating novel metabolites from those unisolated and uncharacterized microorganisms, increasing the possibility of discovering potent antimicrobials, including antifungal agents against A. fumigatus ( Figure 5).
Furthermore, the techniques and methods currently adopted in the isolation and screening of these bioactive compounds require upgrading in order to increase the chances of identifying new broad spectrum antimicrobial agents. For example, mainly focusing on extracellular components from these marine microorganisms seriously limits the global progress in this search. Considering the fact that marine organisms are unique, with special cellular features and components to survive in extreme and peculiar environments, it will be desirable to isolate and test intracellular and membrane-bound polysaccharides, peptides and lipids [103,104].
Moreover, conducting an initial screening of these compounds with predominantly in vitro assays might just be another limitation in this search. It has previously been demonstrated that certain compounds with weak in vitro antimicrobial activities could still be quite effective in in vivo applications [105][106][107][108]. Such compounds could have alternative mechanisms of "conquering the pathogen(s) of interest" beyond biocidal/biostatic activities. Other mechanisms than just the usual growth inhibition of pathogens, such as the modulation of the host immune system or blocking the production of virulence factor(s), are enviable attributes of new compounds that can only be discovered with in vivo assays [107][108][109][110]. Therefore, in vivo screening assays are highly recommended to discover bioactive compounds with broader modes of action.
Using invertebrate models such as Caenorhabditis elegans for a high-throughput in vivo screening has numerous advantages, including the absence of an ethical license requirement, reduced cost, labor and resources, as well as the possibility to simultaneously determine the cytotoxity of bioactive compounds [105,110]. We have recently established a C. elegans-based A. fumigatus infection model [111], making the high-throughput evaluation of the efficacy of bioactive compounds possible. Furthermore, we also discovered that the in vivo efficacy of some antifungal agents was different from what was observed in in vitro screenings, as demonstrated by the different killing modes of amphotericin B and the azole drugs. These therefore provide confidence in high-throughput in vivo assays as the primary screening approach.
Based on the general numbers of bioactive compounds from marine microbes, there are multiple strategies for inducing the production of some essential bioactive metabolites that possess antifungal properties, such as coculturing marine microbes with stimulators (other microorganisms, pathogens/their metabolites) or exposing them to stress conditions. Heavy metal stress could induce the expression of genes that lead to the production of desired metabolites, some of which have also been discovered to possess antimicrobial properties [112][113][114][115][116]. The use of heavy metals in the culture media of certain marine organisms is necessary for inducing the production of some essential metabolites, which may have antifungal properties. Evaluating such antifungal potency on A. fumigatus would really help to broaden the future search and discovery of antimicrobial compounds.
We have therefore suggested cultural approaches in Figure 6 in order to broaden and hasten the search for antifungal agents against A. fumigatus. Six culture approaches, ranging from monocultures to cocultures under different conditions, are suggested to stimulate genes involved in the production of uncommon metabolites, thereby increasing the possibilities of finding novel bioactive compounds that may otherwise not be identified through conventional approaches.

Conclusions
The search for antifungal agents effective against A. fumigatus from the marine habitat has not been all that productive (compared to other similar leading opportunistic pathogens like Candida albicans) for years now. A change of strategy is therefore indispensable and urgent if we must win the raging menacing war against this mold pathogen. Adoption of alternative cultural methods and evaluation of compounds for other treatment mechanisms would help to broaden the horizon of this search and may as well just be the needed breakthrough that we are waiting for.