Bioactivity-Guided Screening of Antimicrobial Secondary Metabolites from Antarctic Cultivable Fungus Acrostalagmus luteoalbus CH-6 Combined with Molecular Networking

With the increasingly serious antimicrobial resistance, discovering novel antibiotics has grown impendency. The Antarctic abundant microbial resources, especially fungi, can produce unique bioactive compounds for adapting to the hostile environment. In this study, three Antarctic fungi, Chrysosporium sp. HSXSD-11-1, Cladosporium sp. HSXSD-12 and Acrostalagmus luteoalbus CH-6, were found to have the potential to produce antimicrobial compounds. Furthermore, the crude extracts of CH-6 displayed the strongest antimicrobial activities with 72.3–84.8% growth inhibition against C. albicans and Aeromonas salmonicida. The secondary metabolites of CH-6 were researched by bioactivity tracking combined with molecular networking and led to the isolation of two new α-pyrones, acrostalapyrones A (1) and B (2), along with one known analog (3), and three known indole diketopiperazines (4–6). The absolute configurations of 1 and 2 were identified through modified Mosher’s method. Compounds 4 and 6 showed strong antimicrobial activities. Remarkably, the antibacterial activity of 6 against A. salmonicida displayed two times higher than that of the positive drug Ciprofloxacin. This is the first report to discover α-pyrones from the genus Acrostalagmus, and the significant antimicrobial activities of 4 and 6 against C. albicans and A. salmonicida. This study further demonstrates the great potential of Antarctic fungi in the development of new compounds and antibiotics.


Identification of the Bioactive Fungi
The three most active fungi were identified by comparing their ITS-rDNA sequences with those in the National Center for Biotechnology Information (NCBI) database, combined with their morphological characteristics. Concretely, the fungus HSXSD-11-1 was identified as Chrysosporium sp. whose 647 bp ITS sequence had 99.52% identity to that of Chrysosporium sp. 2 JC-2013 (HG329729.1) with the query coverage of 95%. The fungus HSXSD-12 was identified as Cladosporium sp. whose 510 bp ITS sequence had 100% identity and 97% query coverage to that of Cladosporium sp. CLAD127 (MK111582.1). The fungus CH-6 was identified as Acrostalagmus luteoalbus whose 574 bp ITS sequence had 100% identity to that of A. luteoalbus J23B1 (MK389477.1) with the query coverage of 95%. The ITS-rDNA sequences of HSXSD-11-1, HSXSD-12, and CH-6 were submitted to GenBank and obtained the accession numbers of MT367260.1, MT367261.1, and MT367202.1, respectively.

Secondary Metabolites Profile Visualization and Annotation by Molecular Networking
The crude extracts of A. luteoalbus CH-6 were subjected to UHPLC-MS/MS analysis to obtain MS 2 data and then the data were converted into .mzXML format to submit into Global Natural Product Social Molecular Networking (GNPS) online workflow for the molecular network of the fungus secondary metabolites profile (Figure 1). The network was visualized by Cytoscape 3.8.0. The molecular network of the fungus secondary metabolites profile contained 615 nodes and 738 edges, suggesting that 615 compounds with different molecular weights were found in the crude extracts of A. luteoalbus CH-6. The 21 yellow nodes were compounds characterized by molecular networking (Table S1). The red nodes were unannotated compounds that might be new compounds according to the result of the molecular networking.

Secondary Metabolites Profile Visualization and Annotation by Molecular Networking
The crude extracts of A. luteoalbus CH-6 were subjected to UHPLC-MS/MS analysis to obtain MS 2 data and then the data were converted into .mzXML format to submit into Global Natural Product Social Molecular Networking (GNPS) online workflow for the molecular network of the fungus secondary metabolites profile (Figure 1). The network was visualized by Cytoscape 3.8.0. The molecular network of the fungus secondary metabolites profile contained 615 nodes and 738 edges, suggesting that 615 compounds with different molecular weights were found in the crude extracts of A. luteoalbus CH-6. The 21 yellow nodes were compounds characterized by molecular networking (Table S1). The red nodes were unannotated compounds that might be new compounds according to the result of the molecular networking. In order to identify the compounds' structures in the first family with the maximum nodes, the literature [13][14][15][16][17][18][19][20][21][22] about secondary metabolites of A. luteoalbus were studied and it was found that thiodiketopiperazine derivatives were the main products of this species, and then annotated 20 analogs in the first family in the molecular network according to the previous studies about thiodiketopiperazine derivatives (Figures 1 and 2) [16,20,[26][27][28][29][30][31][32][33].
The α-pyrone derivatives (Figures 1 and 3) were also annotated in the molecular network according to the literature [34][35][36][37]. The retention time of the mapped α-pyrones in HPLC fingerprint was pointed out with low yield suggesting that it is difficult to isolate α-pyrone derivatives in the extracts of the fungus A. luteoalbus CH-6.
The α-pyrone derivatives (Figures 1 and 3) were also annotated in the molecular network according to the literature [34][35][36][37]. The retention time of the mapped α-pyrones in HPLC fingerprint was pointed out with low yield suggesting that it is difficult to isolate α-pyrone derivatives in the extracts of the fungus A. luteoalbus CH-6.

Structure Elucidations of Isolated Compounds 1-6
The crude extracts obtained by static culture of A. luteoalbus CH-6 showed the strongest antifungal and antibacterial activities. Therefore, they were separated through a bioactivity-guided strategy which led to the isolation of compounds 1-6. Acrostalapyrone A (1) (Figure 4) was obtained as an amorphous powder. Its molecular formula, C14H20O4, was determined by HR-ESI-MS spectrum ( Figure S9), indicating five degrees of unsaturation. Careful analysis of 1 H NMR, 13 C NMR, and HSQC spectra (Figures S3-S5) of 1 revealed five methyl signals, including one oxygenated methyl at δH 3.91 (3H, s), δC 56.3, four methines, including two unsaturated methines at δH 6.46 (1H, dd, 10.3, 1.5 Hz), δC 136.8 and δH 6.13 (1H, s), δC 92.4, and one oxygenated methine at δH 3.72 (1H, p, 6.4 Hz), δC 71.9, and five unsaturated quaternary carbons, including one ester group at δC 165.2 ( Table 4). The five unsaturated quaternary carbons and two unsaturated methines represented four degrees of unsaturation, combined with the whole five degrees of unsaturation, provided the existence of a ring. All of these NMR spectra characters revealed that 1 was a pyrone compound. Further analyzing these data discovered that 1 was very similar to phomenin A [38] and phomapyrone E [39]. The most obvious differences in the NMR data between 1 and phomenin A were the two olefinic carbons signals in phomenin A were substituted by two methines at δH 3.72 (1H, p, 6.4 Hz), δC 71.9, and δH 2.60 (1H, dp, 10.3, 6.7 Hz), δC 41.1 in 1 (Table 4). This was further elucidated by the HMBC correlations from H-9 Me to C-10 and from H-11 to C-9, and the COSY relationships of H-8/H-9, H-9/H-9 Me, H-9/H-10 and H-10/H-11 ( Figure 5). Thus the planar structure of 1 was unambiguously confirmed.

Structure Elucidations of Isolated Compounds 1-6
The crude extracts obtained by static culture of A. luteoalbus CH-6 showed the strongest antifungal and antibacterial activities. Therefore, they were separated through a bioactivityguided strategy which led to the isolation of compounds 1-6. Acrostalapyrone A (1) (Figure 4) was obtained as an amorphous powder. Its molecular formula, C 14 H 20 O 4 , was determined by HR-ESI-MS spectrum ( Figure S9), indicating five degrees of unsaturation. Careful analysis of 1 H NMR, 13 C NMR, and HSQC spectra ( Figures S3-S5) of 1 revealed five methyl signals, including one oxygenated methyl at δ H 3.91 (3H, s), δ C 56.3, four methines, including two unsaturated methines at δ H 6.46 (1H, dd, 10.3, 1.5 Hz), δ C 136.8 and δ H 6.13 (1H, s), δ C 92.4, and one oxygenated methine at δ H 3.72 (1H, p, 6.4 Hz), δ C 71.9, and five unsaturated quaternary carbons, including one ester group at δ C 165.2 ( Table 4). The five unsaturated quaternary carbons and two unsaturated methines represented four degrees of unsaturation, combined with the whole five degrees of unsaturation, provided the existence of a ring. All of these NMR spectra characters revealed that 1 was a pyrone compound. Further analyzing these data discovered that 1 was very similar to phomenin A [38] and phomapyrone E [39]. The most obvious differences in the NMR data between 1 and phomenin A were the two olefinic carbons signals in phomenin A were substituted by two methines at δ H 3.72 (1H, p, 6.4 Hz), δ C 71.9, and δ H 2.60 (1H, dp, 10.3, 6.7 Hz), δ C 41.1 in 1 (Table 4). This was further elucidated by the HMBC correlations from H-9 Me to C-10 and from H-11 to C-9, and the COSY relationships of H-8/H-9, H-9/H-9 Me, H-9/H-10 and H-10/H-11 ( Figure 5). Thus the planar structure of 1 was unambiguously confirmed. Mar. Drugs 2022, 20, x 8 of 16   The relative configurations of 1 were determined by NOESY spectrum ( Figure S8). The NOESY correlation between H-7 Me and H-9 revealed the E configuration of the olefinic bond at C-7 and C-8 ( Figure 6). The cross-peaks of H-8/H-9 Me and H-8/H-11 in the NOESY spectrum proved H-9 Me and H-11 were in the same face. Thus, the relative configurations of 1 were elucidated to be 7E,9S,10R or 7E,9R,10S.     The relative configurations of 1 were determined by NOESY spectrum ( Figure S8). The NOESY correlation between H-7 Me and H-9 revealed the E configuration of the olefinic bond at C-7 and C-8 ( Figure 6). The cross-peaks of H-8/H-9 Me and H-8/H-11 in the NOESY spectrum proved H-9 Me and H-11 were in the same face. Thus, the relative configurations of 1 were elucidated to be 7E,9S,10R or 7E,9R,10S. The absolute configurations of 1 were ascertained by modified Mosher's methods [40,41]. The (S)-and (R)-MTPA esters of 1, 1s, and 1r, were obtained after treatment of 1 with (S)-and (R)-MTPA-Cl, respectively. The 10R configuration of 1 was revealed by the ΔδH(1s-1r) values ΔδH-11 = +0.07, ΔδH-9 = -0.05, ΔδH-9 Me = -0.11, ΔδH-8 = -0.06 (Figure 7), following the Mosher's rules. Therefore, the absolute configurations of 1 were confirmed to be 7E,9S,10R. The α-pyrone analogs were named by the name of the isolated fungi and the structural type of the compounds [38,39], compound 1 was named acrostalapyrone A. Acrostalapyrone B (2) (Figure 4) was gained as an amorphous powder with the molecular formula of C14H20O4 determined by HR-ESI-MS ( Figure S16), which was the same as 1. The 1 H and 13 C NMR data of 2 were very similar to those of 1 (Table 4) indicating that 2 and 1 shared the same plane structure. The obvious differences between the 13 C NMR data (Table 4) of 2 and 1 were the higher field shifts of C-7 (δC 125.7 in 2 vs. δC 126.4 in 1) and C-11 (δC 20.8 in 2 vs. δC 21.3 in 1) which might suggest the different configurations of C-7 and C-10 in 2 and 1.
The relative configurations of 2 were decided by its NOESY spectrum ( Figure S15). The cross peak of H-7 Me and H-9 in NOESY proved the E configuration of the olefinic bond at C-7 and C-8 ( Figure 6) which was the same as 1. The NOESY relationship of H-9 and H-11 indicated that H-9 and H-11 were on the same side ( Figure 6). The NOESY crosspeaks of H-9 Me/H-10 revealed they were on the same side. Thus, the relative configurations of 2 were decided to be 7E,9S,10S or 7E,9R,10R.
The relative configurations of 2 were decided by its NOESY spectrum ( Figure S15). The cross peak of H-7 Me and H-9 in NOESY proved the E configuration of the olefinic bond at C-7 and C-8 ( Figure 6) which was the same as 1. The NOESY relationship of H-9 and H-11 indicated that H-9 and H-11 were on the same side ( Figure 6). The NOESY crosspeaks of H-9 Me/H-10 revealed they were on the same side. Thus, the relative configurations of 2 were decided to be 7E,9S,10S or 7E,9R,10R.
The absolute configurations of 2 were confirmed by modified Mosher's methods [40,41].  Acrostalapyrone B (2) (Figure 4) was gained as an amorphous powder with the molecular formula of C 14 H 20 O 4 determined by HR-ESI-MS ( Figure S16), which was the same as 1. The 1 H and 13 C NMR data of 2 were very similar to those of 1 (Table 4) indicating that 2 and 1 shared the same plane structure. The obvious differences between the 13 C NMR data (Table 4) of 2 and 1 were the higher field shifts of C-7 (δ C 125.7 in 2 vs. δ C 126.4 in 1) and C-11 (δ C 20.8 in 2 vs. δ C 21.3 in 1) which might suggest the different configurations of C-7 and C-10 in 2 and 1.
The relative configurations of 2 were decided by its NOESY spectrum ( Figure S15). The cross peak of H-7 Me and H-9 in NOESY proved the E configuration of the olefinic bond at C-7 and C-8 ( Figure 6) which was the same as 1. The NOESY relationship of H-9 and H-11 indicated that H-9 and H-11 were on the same side ( Figure 6). The NOESY cross-peaks of H-9 Me/H-10 revealed they were on the same side. Thus, the relative configurations of 2 were decided to be 7E,9S,10S or 7E,9R,10R.

Antifungal and Antibacterial Activity Evaluations of Isolated Compounds
All the isolated compounds (1-6) were evaluated for their antifungal and antibacterial activities against one pathomycete C. albicans, four pathogenic bacteria E. coli, S. aureus, B. subtilis, P. aeruginosa, and ten marine fouling bacteria P. fulva, P. aeruginosa, A. salmonicida, A. hydrophila, V. anguillarum, V. harveyi, P. halotolerans, P. angustum, E. cloacae, and E. hormaechei. α-Pyrones 1-3 displayed no obvious antimicrobial activities against all the tested strains, and indole diketopiperazines 4-6 showed antimicrobial activities against a panel of strains (Table 5). Among them, compound 6 exhibited broad-spectrum antimicrobial activities against C. albicans, A. salmonicida, P. halotolerans, P. fulva, and S. aureus with the MIC values range from 3.125 µM to 50 µM (Table 5). Furthermore, compound 6 displayed antibacterial activity two times higher than that of the positive drug Ciprofloxacin against A. salmonicida. Compounds 4 and 6 showed significant antimicrobial activities against C. albicans and A. salmonicida with the MIC values of 3.125-12.5 µM (Table 5).

Isolation and Fermentation of Soil-Derived Fungi from the Fildes Peninsula, Antarctica
The soil samples were collected in ice-free areas (about 10 cm from the surface) of the Fields Peninsula (S62 • 12 , W58 • 58 ) using sterile spatulas and sterilized WhirlPak bags (Sigma-Aldrich, St. Louis, MO, USA), and were transported to the lab in sealed foam package filled with dry ice by airplane, at the Chinese 35th Antarctic expedition in 2019 [44]. The soil samples were incubated in a water bath at 16 • C for 3 min to thaw quickly. In aseptic conditions, 10 g soil sample was mixed thoroughly in 10 mL of sterile distilled water and stood overnight to obtain a bacterial and fungal suspension. The suspension was diluted into 10 −1 , 10 −2 , and 10 −3 with sterile distilled water, and 100 µL of each dilution along with a stock solution was transferred to PDA culture media and evenly dispersed, respectively, each strength of the suspension was repeated three times to incubate at 4 • C, 16 • C, and 28 • C for one to three weeks until no new colonies appear.
Single colonies of fungi were carefully picked into new PDA culture media repeatedly until only one colony grew in the medium. The purified fungi were transferred into cryogenic vials containing potato dextrose water (PDW) culture media with glycerol protection (v/v = 3:1), stored at −80 • C in the State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, China. The isolated fungi were selected with different morphological colonies and cultivated in PDW culture media in two Erlenmeyer flasks (300 mL in each 500 mL flask) at 16 • C, one in static (45 days) condition, another one in shock (14 days) condition.

Extraction and Bioactivities Screening of Fermented Fungi
Each of the fungal fermented culture broth (300 mL) was filtered by two layers of gauze to separate the mycelia from the broth. The mycelia were extracted three times with EtOAc (3 × 200 mL) and then repeatedly extracted with CH 2 Cl 2 -MeOH (v/v, 1:1) three times (3 × 200 mL). The broth was extracted repeatedly with EtOAc (3 × 300 mL) to get the EtOAc layer. All the extracts were combined and then evaporated to dryness under reduced pressure to afford residues.
The bioactive assays were tested in 96 well-plate. Each well contained 198 µL tested strain suspension (2-5 × 10 5 CFU/mL in LB broth) and 2 µL fungal extract (final concentration was 50 µg/mL). Three replicates were performed. The plates were incubated at 37 • C for 24 h, then the OD values were tested at 600 nm in a microplate reader (TriStar 2 S LB 942 Multimode Reader, Berthold Technologies, Germany). The inhibitory rates were calculated according to the following formula: Inhibition rate (%) = (OD DMSO − OD extrat )/OD DMSO × 100

MS 2 Parameters
MS 2 analyses were performed using high-resolution Q-TOF mass spectrometry (Bruker impactHD) coupled with an ESI source with the parameters as followed: positive-ion mode, capillary source voltage at 3500 V, drying-gas flow rate at 4 L/min, drying-gas temperature at 200 • C, and end plate offset voltage at 500 V. MS full scan mode was operated from m/z 50-1500 (100 ms scan time) with a resolution of 40,000 at m/z 1222.

Extraction and Isolation of Compounds 1-6 from Acrostalagmus luteoalbus CH-6
The fungal strain Acrostalagmus luteoalbus CH-6 was fermented in a rice culture medium in 200 Erlenmeyer flasks (250 g rice and 350 mL water in each 1000 mL flask) at 16 • C in an air-conditioned room for 60 days. Rice culture medium was used for its eutrophy, simple preparation, low cost, convenience, and ease to obtain. On the other hand, the crude extracts of fungus A. luteoalbus CH-6 cultured in rice medium exhibited stronger antimicrobial activities against C. albicans and A. salmonicida. The fermented culture of A. luteoalbus CH-6 (50 kg) was extracted three times with EtOAc (3 × 4000 mL) and then repeatedly extracted with CH 2 Cl 2 -MeOH (v/v, 1:1) three times (3 × 4000 mL). All the extracts were combined and then evaporated to dryness under reduced pressure to afford a residue (376 g). The residue was subjected to vacuum liquid chromatography (VLC) on silica gel using step gradient elution with EtOAc-petroleum ether (PE) (0-100%) and then with MeOH-EtOAc (0-100%) to afford eight fractions (Fr.1-Fr.8). All the eight fractions (Fr.1-Fr.8) were evaluated for their antimicrobial activities against C. albicans and A. salmonicida and the results showed that the primary bioactive compounds produced by the fungus A. luteoalbus CH-6 were focused on Fr.3 and Fr.4. Through the analysis of HPLC-DAD-UV fingerprints ( Figure S33) and antimicrobial activity evaluations of Fr.1-Fr.8 (Table  S3) (Table S3). Fr.3.5.2 was purified by using semi-preparative HPLC on an ODS column (Kromasil C 18 , 250 × 10 mm, 5 µm, evaluated using the 2-fold serial-dilution method. The concentrations of the compounds ranged from 100 µM to 0.78125 µM. The other steps were the same as the method of primary screening.

Conclusions
In summary, three antimicrobial fungi, Chrysosporium sp. HSXSD-11-1, Cladosporium sp. HSXSD-12, and Acrostalagmus luteoalbus CH-6, were discovered from the soil samples of the Fildes Peninsula, Antarctica. Bioassay-guided searching of antimicrobial secondary metabolites of A. luteoalbus CH-6, combined with molecular networking, led to the isolation of two new α-pyrones, acrostalapyrones A (1) and B (2), and one known analog, multiforisin G (3), as well as three known indole diketopiperazines, luteoalbusin A (4), gliocladine C (5), and T988 C (6). Compounds 4 and 6 showed significant antimicrobial activities against C. albicans and A. salmonicida. In particular, the antibacterial activity against A. salmonicida of 6 displayed two times higher than that of the positive drug Ciprofloxacin. This is the first time to find α-pyrones in the fungal genus Acrostalagmus and the first report to discover significant antimicrobial activities of compounds 4 and 6 against C. albicans and A. salmonicida. This study further demonstrates the great potential of Antarctic fungi in the development of new compounds and antibiotics.