*Article* **Secondary Metabolite Variation and Bioactivities of Two Marine** *Aspergillus* **Strains in Static Co-Culture Investigated by Molecular Network Analysis and Multiple Database Mining Based on LC-PDA-MS/MS**

**Yuan Wang 1,2, Evgenia Glukhov <sup>3</sup> , Yifan He <sup>3</sup> , Yayue Liu 1,2, Longjian Zhou 1,2 , Xiaoxiang Ma 1,2 , Xueqiong Hu <sup>1</sup> , Pengzhi Hong 1,2, William H. Gerwick <sup>3</sup> and Yi Zhang 1,2,3,\***


**Abstract:** Co-culture is known as an efficient way to explore the metabolic potential of fungal strains for new antibiotics and other therapeutic agents that could counter emerging health issues. To study the effect of co-culture on the secondary metabolites and bioactivities of two marine strains, *Aspergillus terreus* C23-3 and *Aspergillus. unguis* DLEP2008001, they were co-cultured in live or inactivated forms successively or simultaneously. The mycelial morphology and high-performance thin layer chromatography (HPTLC) including bioautography of the fermentation extracts were recorded. Furthermore, the agar cup-plate method was used to compare the antimicrobial activity of the extracts. Based on the above, liquid chromatography-photodiode array-tandem mass spectrometry (LC-PDA-MS/MS) together with Global Natural Products Social molecular networking (GNPS) and multiple natural products database mining were used to further analyze their secondary metabolite variations. The comprehensive results showed the following trends: (1) The strain first inoculated will strongly inhibit the growth and metabolism of the latter inoculated one; (2) Autoclaved *A. unguis* exerted a strong inducing effect on later inoculated *A. terreus*, while the autoclaved *A. terreus* showed high stability of its metabolites and still potently suppressed the growth and metabolism of *A. unguis*; (3) When the two strains are inoculated simultaneously, they both grow and produce metabolites; however, the *A. terreus* seemed to be more strongly induced by live *A. unguis* and this inducing effect surpassed that of the autoclaved *A. unguis*. Under some of the conditions, the extracts showed higher antimicrobial activity than the axenic cultures. Totally, *A. unguis* was negative in response but potent in stimulating its rival while *A. terreus* had the opposite effect. Fifteen MS detectable and/or UV active peaks showed different yields in co-cultures vs. the corresponding axenic culture. GNPS analysis assisted by multiple natural products databases mining (PubChem, Dictionary of Natural Products, NPASS, etc.) gave reasonable annotations for some of these peaks, including antimicrobial compounds such as unguisin A, lovastatin, and nidulin. However, some of the peaks were correlated with antagonistic properties and remain as possible novel compounds without mass or UV matching hits from any database. It is intriguing that the two strains both synthesize chemical 'weapons' for antagonism, and that these are upregulated when needed in competitive co-culture environment. At the same time, compounds not useful in this antagonistic setting are downregulated in their expression. Some of the natural products produced during antagonism are unknown chlorinated

**Citation:** Wang, Y.; Glukhov, E.; He, Y.; Liu, Y.; Zhou, L.; Ma, X.; Hu, X.; Hong, P.; Gerwick, W.H.; Zhang, Y. Secondary Metabolite Variation and Bioactivities of Two Marine *Aspergillus* Strains in Static Co-Culture Investigated by Molecular Network Analysis and Multiple Database Mining Based on LC-PDA-MS/MS. *Antibiotics* **2022**, *11*, 513. https://doi.org/10.3390/ tantibiotics11040513

Academic Editors: Fuhang Song and Yunjiang Feng

Received: 19 February 2022 Accepted: 6 April 2022 Published: 12 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

metabolites and deserve further study for their antimicrobial properties. In summary, this study disclosed the different responses of two *Aspergillus* strains in co-culture, revealed their metabolic variation, and displayed new opportunities for antibiotic discovery.

**Keywords:** *Aspergillus terreus*; *Aspergillus unguis*; co-culture; antimicrobial activity; LC-PDA-MS/MS; molecular network; database mining

#### **1. Introduction**

The ocean supports an amazing variety of marine life and is a crucial part of the biosphere. Marine organisms, including microbes, have developed complex metabolic mechanisms to adapt themselves to the unique environment of high salinity, high pressure, low oxygen, and oligotrophy. As a result, secondary metabolites (SMs) with novel structures and rich activities are produced and provide a rich source of drug lead compounds [1]. Although medical sciences have made significant progress, infectious diseases caused by bacteria, fungi, and viruses still pose a substantial threat to public health. Due to the development of antibiotic resistance, finding new antibiotics remains an essential task for scientists worldwide. Nevertheless, most of the current antibacterial agents derived from natural products were isolated from terrestrial sources, while marine organisms are still primarily untapped resources for new biologically active natural products, and especially antibiotics [2].

Previous studies have shown that fungi contain many diverse biosynthetic gene clusters that encode for secondary metabolites, but under artificial culture conditions in the laboratory, most fungal functional gene clusters are silent (i.e., not expressed). The methods for enriching the diversity of expressed fungal metabolites include changing the composition of the medium, changing the environmental conditions, adding epigenetic modifiers, and co-cultivating with other fungi or bacteria [3–5]. Specifically, the co-cultivation of microorganisms from different sources often creates competition and antagonism. To compete for the limited natural resources in such an environment, or for living space or to maintain information transmission between species, the microbes often produce secondary metabolites that are not produced when cultured separately [6].

Mass spectrometry (MS)-based metabolomics is increasingly playing an important role in efficient natural products studies. These approaches enable the accurate offline and online comparison of constituent differences among samples including big data samples. However, these data sets are often too large for manual analysis as more than 1000 MS/MS spectra can be collected from just one extract sample. Global Natural Products Social molecular networking (GNPS) is a data-driven open platform for the storage, analysis, and dissemination of MS/MS spectra. It provides the ability to visualize data sets from different users and compare these with all publicly available reference spectra to annotate known molecules and discover putative analogs [7]. For example, Oppong-Danquah. et al. used a GNPS molecular networking-based screening method to annotate metabolites with crop protection activity in co-cultures of several marine fungi, significantly improving the efficiency of discovery and identification of trace novel natural products [8]. GNPS is a continuously developing platform for accurate dereplication and annotation tasks, and thus is best complemented with other approaches at the present time.

In preliminary studies, our laboratory obtained a marine *Aspergillus terreus* strain C23-3 from a coral collected in Xuwen Natural Reserve of South China Sea and a marine *A. unguis* strain DLEP2008001 from a seaweed collected at the intertidal zone of Dalian City by the Yellow Sea of Northern China. Intriguingly, our previous research and the reports from other groups indicated that both these two species/strains can produce potent antibiotics as well as other bioactive compounds. For example, *A. terreus* produces butyrolactones showing antibacterial, antitumor, antioxidant, antiviral, enzymes (glucosidase, glucuronidase, and cyclin-dependent protein kinase 5) inhibitory, neuroprotective,

anti(-neuro-)inflammatory, and axonal growth promoting activities [9–14], as well as lipid lowering lovastatins and acetylcholinesterase (AChE) inhibitory territrems [15,16]. As for *A. unguis*, it was known to produce halogenated and non-halogenated depsidones which were reported to possess antibacterial, antifungal, brine shrimp larvacidal, enzyme inhibitory (AChE and aromatase), diphenyl-picryl hydrazyl (DPPH) free radical scavenging, and neuroprotective activities [17–25].

Because both strains are producers of antibiotics and neuroactive agents, it was appealing to investigate the effect of co-culture conditions on expression of their secondary metabolites, as this might result in the discovery of new antibiotics or anti-neurodegenerative agents. In this paper, we investigated the high-performance thin layer chromatographic (HPTLC) profiles, bioactivities, LC-MS/MS based GNPS molecular networking, and multiple natural product database mining of secondary metabolites deriving from co-culturing of these two marine fungi.

#### **2. Results**

#### *2.1. Morphological Comparison*

The two strains *A. unguis* and *A. terreus* were statically cultivated for a total of 28 days (in one stage or two stages) in seawater potato sucrose broth under 7 experimental condition groups, including: G1) *A. unguis* axenically for 28 days (abbreviated as axU); G2) *A. terreus* axenically for 28 days (axT); G3) *A. unguis* 7-day culture-inactivation + live *A. terreus* for the following 21 days (iacU-livT); G4) Live *A. unguis* 7-day culture + live *A. terreus* for the following 21 days (livU-livT); G5) live *A. unguis* and live *A. terreus* inoculated simultaneously (livU/livT) and co-cultivated for 28 days; G6) *A. terreus* 7-day culture-inactivation + live *A. unguis* for the following 21 days (iacT-livU); G7) Live *A. terreus* 7-day culture + live *A. unguis* for the following 21 days (livT-livU) (see details of the culture experiments in Section 4.2.1).

When *A. unguis* grows axenically, it initially forms scattered bright yellow colonies and then merges into a dark brown mycoderm (Figure 1G1). When *A. terreus* grows alone, it initially forms white colonies and then expands to form an off-white to brown mycoderm (Figure 1G2). If *A. terreus* is inoculated on the autoclaved 7-day culture of *A. unguis*, it still grows but just as small scattered off-white colonies on the dead mycoderm of *A. terreus* without forming its own continuous mycoderm (Figure 1G3). If *A. terreus* is inoculated on the live 7-day culture of *A. unguis*, no obvious growth of *A. terreus* is observed (Figure 1G4). When *A. unguis* and *A. terreus* are inoculated simultaneously, two types of mycoderms are observed: dark brown and off-white (Figure 1G5). When *A. unguis* is inoculated on the autoclaved 7-day culture of *A. terreus*, it does not grow as well as the axenic *A. unguis* culture but still forms its own complete mycoderm on the surface of the dead mycoderm of *A. terreus* (Figure 1G6). However, if *A. unguis* is inoculated on the live 7-day culture of *A. terreus*, the newcomers' growth is not obvious (Figure 1G7). Comparing the different culture experiments, it was found that the early inoculated fungus (even if autoclaved) will inhibit the growth of the late inoculated one, and the live fungus showed stronger inhibition than the inactivated one.

**Figure 1.** The morphology of axenic cultures and co-cultures in different experiments (28 days in total). (**G1**–**G7**), respectively, represent: axU (**G1**), axT (**G2**), iacU-livT (**G3**), livU-livT (**G4**), livU/livT (**G5**), iacT-livU (**G6**), livT-livU (**G7**).

#### *2.2. Comparison of HPTLC Fingerprints*

The profiles of secondary metabolites including their antioxidant as well as anti-AChE constituents were demonstrated using HPTLC images that were observed under 254 nm and 365 nm, colored by anisaldehyde reagent and potassium ferricyanide-ferric chloride (PFFC) reagent, and revealed by DPPH free radical scavenging and AChE inhibitory bioautographies, respectively. These HPTLC images revealed the variation between axenic cultures and co-cultures in different ways, including some dramatical changes.

The UV images (under 254 nm and 365 nm) clearly showed rich secondary metabolic profiles from the cultures. In the profile of axenic *A. unguis* (lane 1 in Figure 2A under 254 nm), the big dark spot with Rf value of 0.70 was judged to be comprised of depsidones according to our previous study on this strain [18]. For axenic *A. terreus*, the dark spot with Rf value of 0.28 was recognized from previous work as butyrolactone I [11]. These annotations were also supported by the LC-PDA-MS/MS analysis as described below in Section 2.4.

**Figure 2.** HPTLC fingerprints of the axenic and co-cultural extracts. (**A**) is the UV images of experiments G1–G7 under 254 nm (the sample numbers were marked with pencil below the starting line). (**B**) is the UV images of G1–G7 under 365 nm. (**C**) is the image of sulfuric acid-anisaldehyde colorized plate of G1–G7. (**D**) is the image of potassium ferricyanide-ferric chloride (PFFC) colorized plate of G1–G7. (**E**) is the DPPH free radical scavenging autographic image of G1–G7. (**F**) is acetylcholinesterase inhibitory bioautographic image of G1–G7. The developing agent was chloroform:methanol = 20:1 (*v*/*v*). The rulers beside the TLC plate are taken as references for Rf value calculation. The yellow or red arrows mark the new metabolites produced only under co-cultural conditions.

In the experiment G3 (iacU-livT), some *A. terreus* metabolites disappeared including the spots at Rf 0.43 (orange fluorescence), Rf 0.38 (red fluorescence), and Rf 0.17–0.30 (dark blue, including butyrolactone I). However, other putative *A. terreus* metabolites were enhanced in their production, including white fluorescent spots at Rf 0.84 and Rf 0.68. Moreover, several new constituents appeared in this co-culture including fluorescent spots at Rf 0.76 (light orange), Rf 0.58 (white), and Rf 0.20 (white) as marked with the yellow arrows (Figure 2B under 365 nm). Remarkably, the typical *A. unguis* depsidone metabolites still appeared in this co-culture (Figure 2A under 254 nm), indicating their thermostability against autoclaving.

In the experiment G4 (livU-livT, inoculated in tandem), the UV images were basically the same as those of the axenic *A. unguis*, indicating that the later inoculated *A. terreus* was extremely suppressed in its growth and production of metabolites. This is consistent with the morphological observation described above.

In experiment G5 (livU/livT, inoculated simultaneously), the UV images greatly differed from those of G3 and G4, but closely resembled those of G2 (the axenic *A. terreus*). However, some *A. terreus* products were produced in lower yields such as the spot with Rf 0.38 (red fluorescence), or even vanished such as the one with Rf 0.43 (orange fluorescence). In contrast, some spots, like the white fluorescent spots with Rf 0.84, 0.67, 0.32, and 0.08, were significantly enhanced to a much higher extent than in co-culture G3. As for *A. unguis* metabolites, only a small quantity of depsidone metabolites (Rf 0.67) was observed under 254 nm. This situation agreed with the growth advantage of *A. terreus* vs. *A. unguis* in Figure 1G5.

In the experiment G6 (iacT-livU), the UV image under 365 nm was similar to that of axenic *A. terreus*, suggesting that the *A. terreus* metabolites were quite thermotolerant against autoclaving and were stable over a three-week period. The depsidones of *A. unguis* were present but with much lower yield compared to the axenic *A. unguis* culture. Therefore, even the autoclaved *A. terreus* can remarkably inhibit the growth or metabolism of *A. unguis*.

Likewise, in experiment G7 (livT-livU, inoculated in tandem), the UV image displayed almost identical features with the axenic *A. terreus* cultures; however, the depsidones from *A. unguis* could barely be observed.

The results of anisaldehyde and PFFC colorization (Figure 2C,D) were consistent with the UV findings. Additionally, they revealed the production of new metabolite during the co-cultivation experiments. For example, the following were new compounds: blue gray spot at Rf 0.58 in experiment G3, gray spot at Rf 0.82 in G4 (both with anisaldehyde detection), brown spot at Rf 0.27 and brownish spot at Rf 0.40 (both with PFFC detection).

The two bioautographies revealed the antioxidant and anti-AChE constituents in the different culture experiments (Figure 2E,F). The depsidones showed antioxidant and anti-AChE activities, while butyrolactone I showed antioxidant activity. Their variation in amounts (Figure 2A) were partially reflected in the changes of the bioactive spots in these bioautography experiments. Nevertheless, some minute new anti-AChE spots (Rf 0.32 and 0.36) were also observed in co-culture G3, and differences in highly polar constituents remaining at the point of application cannot be excluded because the mobile phase (chloroform:methanol = 20:1 (*v*/*v*)) was not polar enough to mobilize them in the chromatogram.

Generally, the HPTLC suggested the following trends: (1) the strain firstly inoculated will strongly inhibit the growth and metabolism of the later inoculated one, (2) the autoclaved *A. unguis* exerted a strong inducing effect on the later inoculated *A. terreus*, while the autoclaved *A. terreus* showed high stability of its metabolites and still potently suppressed the growth and metabolism of *A. unguis*, (3) when the two strain were inoculated simultaneously, they both grew and produced metabolites. However, the *A. terreus* seemed to be more strongly induced by live *A. unguis* and this inducing effect surpassed that of the autoclaved *A. unguis*. Finally, *A. unguis* was negative in response and agonism but potent in stimulating its rival while *A. terreus* had the opposite effect.

#### *2.3. Antimicrobial Activity*

Based on the above preliminary co-cultural product HPTLC analysis, the antimicrobial activities of the extracts were further tested against several indicator strains, including Methicillin-resistant *Staphylococcus aureus* (MRSA), *Bacillus subtilis*, *Pseudomonas aeruginosa*, *Vibro parahemolyticus*, *V. alginolyticus*, *Shewanella putrefaciens*, *Yersinia pseudotuberculosis*, and *Candida albicans*. The results are shown in Table 1 and the representative photos below in Figure 3.


**Table 1.** The total extract amounts and antimicrobial activities from experiments G1–G7, which were measured using the Oxford Cup method (dosage: 200 mL/well, concentration = 1 mg/mL, concentration for both controls = 0.1 mg/mL, *n* = 4).
