Antibacterial Secondary Metabolites from Marine-Derived Fungus Aspergillus sp. IMCASMF180035

Four new secondary metabolites, including one spiro[anthracenone-xanthene] derivative aspergiloxathene A (1), one penicillide analogue, Δ2′-1′-dehydropenicillide (2), and two new phthalide derivatives, 5-methyl-3-methoxyepicoccone (3) and 7-carboxy-4-hydroxy-6-methoxy-5-methylphthalide (4), together with four known compounds, yicathin C (5), dehydropenicillide (6), 3-methoxyepicoccone (7), 4-hydroxy-6-methoxy-5-methylphthalide (8), were identified from the marine-derived fungus Aspergillus sp. IMCASMF180035. Their structures were determined by spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HRESIMS), 1D and 2D nuclear magnetic resonance (NMR) techniques. Compound 1 was identified as the first jointed molecule by xanthene and anthracenone moieties possessing an unprecedented carbon skeleton with spiro-ring system. All compounds were evaluated activities against Staphylococcus aureus, methicillin resistant S. aureus (MRSA), Escherichia coli, Escherichia faecium, Pseudomonas aeruginosa, and Helicobacter pylori. Compound 1 showed significant inhibitory effects against S. aureus and MRSA, with minimum inhibitory concentration (MIC) values of 5.60 and 22.40 µM. Compounds 2 and 6 exhibited potent antibacterial activities against H. pylori, with MIC values of 21.73 and 21.61 µM, respectively.


Introduction
Overuse of antibiotics has led to the emergence and maintenance of drug resistance. The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species) pathogens are responsible for a variety of infectious diseases with a wide range of drug resistance to current clinical drugs [1]; therefore, there is urgent need to develop novel antibiotics [2,3]. Natural products have historically played an important role in the development of novel antibacterial agents [4]. Natural products characterized from marine-derived organisms are considered more and more important for drug development [5][6][7]. In recent years, more than 1000 new compounds were identified from organisms living in marine habitats per annum [8][9][10], which suggests the great potential of discovering new chemical entries from marine-derived organisms.

Culture of Fungus and Isolation of Compounds
Aspergillus sp. IMCASMF180035 was inoculated into 1 L flasks with rice solid medium and incubated stationary at 28 °C for 30 days. The cultures and medium were extracted by EtOAc:MeOH and followed by a series of purification methods, such as partition, reduced normal phase silica chromatography, Sephadex LH-20 chromatography, and high-performance liquid chromatography according to our previous report [11,12] to yield compounds 1-8.

Structure Elucidation
Compound 1 was isolated as a light yellow powder. The molecular formula of 1 was determined to be C30H22O11 based on the high-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum (m/z [M + H] + 559.1235, calcd for C30H22O11 + , 559.1235), accounting for 20 degrees of unsaturation (Supplementary Figure S1). The 1 H NMR and ¹H-¹H correlation spectroscopy data of 1 (Table 1

Culture of Fungus and Isolation of Compounds
Aspergillus sp. IMCASMF180035 was inoculated into 1 L flasks with rice solid medium and incubated stationary at 28 • C for 30 days. The cultures and medium were extracted by EtOAc:MeOH and followed by a series of purification methods, such as partition, reduced normal phase silica chromatography, Sephadex LH-20 chromatography, and highperformance liquid chromatography according to our previous report [11,12] to yield compounds 1-8.

Biological Activity
All of the compounds were subjected to antibacterial assays against S. aureus ATCC 25923, methicillin-resistant S. aureus USA300, E. coli ATCC 11775, E. faecium ATCC 19434, P. aeruginosa PAO1, and H. pylori G27. Compound 1 exhibited significant antibacterial activities against S. aureus and methicillin-resistant S. aureus (MRSA), with MIC values of 5.60 and 22.40 mM, respectively (positive control, vancomycin, MIC = 0.35 mM). The compounds containing xanthone or anthracenone moieties showed antibacterial activities against a panel of pathogens [13,30,31]. Compound 1 was the first natural product containing a specific carbon skeleton with a spiro-ring system of xanthene and anthracenone, which may offer the antibacterial activities against drug-sensitive and -resistant S. aureus strains. Compounds 2 and 6 exhibited potent antibacterial activities against H. pylori, with MIC values of 21.73 and 21.61 mM, respectively (positive control, metronidazole, MIC = 11.68 mM).

General Experimental Procedures
Optical rotations ([α] D ) were recorded on an Anton Paar MCP 200 Modular Circular Polarimeter (Austria) in a 100 × 2 mm cell at 22 • C. One-dimensional and two-dimensional NMR spectra were measured at 25 • C using a Bruker Avance 500 spectrometer with residual solvent peaks as references (DMSO-d 6 : δ H 2.50, δ C 39.52; CDCl 3 : δ H 7.26, δ C 77.16). high-resolution electrospray ionization mass spectrometry (HRESIMS) measurements were obtained on an Accurate-Mass-Q-TOF LC/MS 6520 instrument (Santa Clara, CA, USA) in positive ion mode. HPLC was performed using an Agilent 1200 Series HPLC System equipped with a diode array detector, a fraction collector, and an Agilent ZORBAX Eclipse XDB-C8 column (250 × 9.4 mm, 5 µm).

Microbial Material
Strain IMCAS180035 was isolated from a mud sample collected from the intertidal zones of the Yellow Sea in Qingdao, China, and grown on a potato dextrose agar plate at 28 • C. This strain was identified as Aspergillus sp. based on gene sequence analysis of the internal transcribed spacer (ITS) (Supplementary Figure S29)

Antibacterial Activity Assays
The microbial inhibition assays were carried out according to the Antimicrobial Susceptibility Testing Standards outlined by the Clinical and Laboratory Standards Institute document M07-A7 (CLSI) [32] by using a penal of pathogens of S. aureus ATCC 25923, MRSA USA300, E. coli ATCC 11775, E. faecium ATCC 19434, P. aeruginosa PAO1, and H. pylori G27. Briefly, the bacteria (S. aureus, MRSA, E. coli, E. faecium, and P. aeruginosa) were taken out from glycerol stocks and inoculated on lysogeny broth (LB) agar plate and cultured overnight at 37 • C. Then, single colonies were picked from the agar plates and adjusted to approximately 10 4 colony-forming unit (CFU)/mL with Mueller-Hinton Broth. Two microliters of 2-fold serial dilution of each compound (in DMSO) were added to each row on a 96-well microplate containing 100 µL of bacterial suspension in each well. Vancomycin and ciprofloxacin were used as positive controls, and DMSO was used as a negative control. The 96-well plate was incubated at 37 • C aerobically for 24 h. Anti-H. pylori (G27) was prepared in a 96-well microtiter plate containing 100 µL of Brain Heart Infusion (BHI) broth supplemented with 10% fetal calf serum (FCS). Metronidazole was used as a positive control. The liquid culture of 2-day-old H. pylori was diluted 10 times in BHI broth to yield a final concentration of 5 × 10 5 to 1 × 10 6 CFU/mL. Two microliters of 2-fold serial dilution of each compound (in DMSO) were added into each well of the testing plates. The testing plates were incubated in a microaerophilic atmosphere at 37 • C for 3 days. The MIC was determined to be the lowest concentration, which resulted in no visible turbidity [33,34].