New Polyketides from a Hydrothermal Vent Sediment Fungus Trichoderma sp. JWM29-10-1 and Their Antimicrobial Effects

Marine fungi-derived secondary metabolites are still an important source for the discovery of potential antimicrobial agents. Here, five new polyketides (1, 2, and 6–8) and seven known compounds (3–5 and 9–12) were obtained from the culture of the marine-derived fungus Trichoderma sp. JWM29-10-1. Their structures were identified by extensive spectrographic data analyses, including 1D and 2D NMR, UV, IR, and HR-ESI-MS. Further, the absolute configurations of new compounds were determined by circular dichroism (CD) spectrum and alkali-hydrolysis in combination with the in situ dimolybdenum CD method. Subsequently, the antimicrobial effects of these isolated compounds were assessed by examining the minimal inhibition concentration (MIC) with the broth microdilution assay. Compounds 1 and 2 exhibited potent antimicrobial activity against Helicobacter pylori, including multidrug-resistant strains, with MIC range values of 2–8 µg/mL. Moreover, compound 1 showed significant inhibitory effects on the growth of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, and vancomycin-resistant Enterococcus faecium, which greatly threaten human health. This study demonstrates that chromone derivatives 1–2, especially for 1, could be potential lead compounds for the development of new antimicrobial agents and provides insight for future medicinal chemistry research.


Introduction
Marine fungi have the ability to produce biologically active lead compounds due to their special living environment. Increasing numbers of marine natural products (MNP) from marine fungi have been newly discovered in recent years [1,2]. Trichoderma sp. fungus is a filamentous fungus that mainly exists in marine animals, plants, and sediments attached to the seafloor [3,4]. Numerous chemical and pharmacological investigations have proved that Trichoderma sp. strains can produce abundant secondary metabolites, which exert potential anti-phytopathogenic, insecticidal, cytotoxic, antibacterial, and antioxidant activities, etc. [4,5] Polyketides, an important type of secondary metabolites from Trichoderma sp., were reported with vital antibacterial and anti-phytopathogenic effects [6]. Khamthong et al. reported the isolation of two new polyketides from Trichoderma aureoviride PSU-F95 and discovered one compound exhibited a certain antibacterial activity against Methicillinresistant Staphylococcus aureus (MRSA) [6]. As infectious diseases seriously threaten human health due to the frequent occurrence of antibiotic resistance [7], polyketides have attracted reoviride PSU-F95 and discovered one compound exhibited a certain antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) [6]. As infectious diseases seri ously threaten human health due to the frequent occurrence of antibiotic resistance [7] polyketides have attracted significant attention due to their diverse biological effects, es pecially for their remarkable antimicrobial effects [8].
As part of our ongoing search for antimicrobial bioactive compounds from marin fungi, twelve polyketides, including five new compounds (1-2 and 6-8) and seven known ones (3-5 and 9-12), were isolated from the ethyl acetate (EtOAc) extract of the culture o fungus Trichoderma sp. JWM29-10-1. Here we described the isolation and chemical char acterization of these isolated compounds. Meantime, the antimicrobial effects of thes compounds were evaluated, and their preliminary structure-activity relationship (SAR was also discussed.
Compound 8 was obtained as a pale-yellow oil. Its molecular formula was elucidated as C 25 Figure 2). The absolute configuration of 8,9-diols in 8 was also determined by alkali-hydrolysis followed by in situ dimolybdenum CD method. The negative Cotton effects of the complex formed by 8A with Mo 2 (OAc) 4 at 310 and 400 nm inferred the 8R and 9S configurations in 8. Thus, the configurations of 8 were determined as 4S, 5S, 6R, 8R, 9S, 10R, 13S, and 14R. Finally, the structure of 8 was identified and named Tandyukisin I.

Antimicrobial Effects of Compounds 1-12
The antimicrobial effects of compounds 1-12 were evaluated by the broth microdilution assay. Results showed that compounds 1 and 2 have efficient antibacterial activities against Helicobacter pylori standard strains and clinical isolates, including three multidrug-resistant strains, with minimal inhibition concentration (MIC) values ranging from 2-8 µg/mL (Table 3). Interestingly, compound 1 also exhibited significant in-hibitory effects on the growth of Gram-positive pathogens, including Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE) and Enterococcus faecalis with MIC values of 2 to 16 µg/mL (Table 3 and Figure 5). In addition, compound 1 exerted moderate antimicrobial activity against the important fungal pathogen Candida albicans and Aspergillus fumigatus with MIC values of 16 and 64 µg/mL, indicating that compound 1 has broad-spectrum antimicrobial activity. Preliminary structure-activity relationship (SAR) analysis revealed that the double bond at C-2 and C-3 of the chromone core structure might be unfavorable for its anti-H. pylori effects based on our limited results since compound 2 showed a stronger inhibitory effect than 3. To our surprise, the introduction of a 3-methyl-2-pentenedioic acid side chain at C-13 could not only dramatically increase the anti-H. pylori activities of chromone derivatives, but also broad the antimicrobial spectrum from Gram-negative to Gram-positive bacteria and fungi. compound 1 has broad-spectrum antimicrobial activity. Preliminary structure-activity relationship (SAR) analysis revealed that the double bond at C-2 and C-3 of the chromone core structure might be unfavorable for its anti-H. pylori effects based on our limited results since compound 2 showed a stronger inhibitory effect than 3. To our surprise, the introduction of a 3-methyl-2-pentenedioic acid side chain at C-13 could not only dramatically increase the anti-H. pylori activities of chromone derivatives, but also broad the antimicrobial spectrum from Gram-negative to Gram-positive bacteria and fungi.

Conclusions and Discussion
Marine fungal secondary metabolites have played a tremendous role in the discovery of anti-infectious drugs in the last 50 years [20][21][22]. In this study, five chromone derivatives (1)(2)(3)(4)(5) and seven decalin derivatives (6)(7)(8)(9)(10)(11)(12), including five new compounds (1, 2, and 6-8), were isolated from the culture of Trichoderma sp. JWM29-10-1. Chromone derivatives with 4-oxo-4H-1-benzopyran core scaffold represent a class of polyketides that were widely distributed in Trichoderma sp. and were reported with antifungal and cytotoxic effects. However, the systemic antimicrobial effect evaluation against bacteria and fungi and SAR were not performed up to now. Here compounds 1-2 exhibited potent antimicrobial effects with MIC values ranging from 2-16 µg/mL. Interestingly, compound 1 exhibited broad antimicrobial effects, especially for killing multidrug-resistant H. pylori. In addition, our study revealed that compound 1 showed significant inhibitory effects on the growth of MRSA and VRE, two of the major causes of community-acquired and hospital-acquired infections that threaten human health.
The rapid spread of multidrug-resistant (MDR) bacteria is a major concern for global public health. This threat is aggravated by an increasingly depleted antibiotic pipeline [23,24], with alarmingly few new classes of antibiotics introduced into clinical use over the past decades. In 2017, the World Health Organization (WHO) released a list of antibioticresistant "priority pathogens"-a catalogue of 12 bacterial species in urgent need of new antibiotics [25]. In this list, clarithromycin-resistant H. pylori, MRSA and VRE were ranked as 'high" priority pathogens. In this study, compound 1 displayed a potent killing activity against these three important bacterial pathogens. To sum up, our studies shed light on the discovery of novel broad-spectrum antimicrobial agents and provide insight for future medicinal chemistry research.

Fungal Material
The fungal strain JWM29-10-1 was collected and separated from hydrothermal vent sediments of Kueishantao, Taiwan, China and identified as Trichoderma sp. according to the morphological characteristics and the 18s rDNA sequence (OP501833), which is 99.9% similar to that of Trichoderma reesei (CBS999.97). The strain was preserved in Ocean College, Zhejiang University, Zhejiang, China.

Fermentation and Extraction
Strain Trichoderma sp. JWM29-10-1 was inoculated on a PDA agar plate, which consisted of 200 g potatoes, 20 g glucose, and 20 g agar in 1 L ddH 2 O. The spores from the agar plate were transferred into a triangular flask containing 100 mL PDA liquid medium and put in a constant temperature shaking incubator for 5 days (28 • C, 180 rpm/min) to obtain 1000 mL of seed culture solution (100 mL × 10). Then 20 mL of seed culture solution was inoculated to a solid rice medium composed of 100 g rice in 150 mL ddH 2 O. A total of 2.5 kg of large-scale fermentation was executed in a solid rice medium and cultured at room temperature for 45 days. The fermentation broth was extracted with EtOAc, and the filtrate was concentrated to dryness under reduced pressure to get crude extracts (30.0 g).

Absolute Configuration Determination of Compounds 6-8
To a solution of compound 6 (16 mg) in 2.5 mL, MeOH was added, 2 mL of aqueous NaOH (1.0 M). Subsequently, the mixture was reacted at room temperature for 24 h. Then, the reaction mixture was extracted with MeOH thrice, and the organic layer was dried under reduced pressure to afford 6A (11.5 mg). Following the same procedure, 7 (4.1 mg) and 8 (4.9 mg) were hydrolyzed with 0.3 M NaOH (aq.) to produce 7A (2.9 mg) and 8A (3.5 mg), respectively. Then, a mixture of 6A, 7A, or 8A with dimolybdenum tetraacetate [Mo 2 (OAc) 4 ] (1:1.2) in DMSO solution was kept to react for 30 min to form the chiral complexes. Then, the CD spectra of the complexes were measured.

Antimicrobial Assays
Antimicrobial assays were assessed by the broth microdilution assay following the previous literature [26][27][28] according to CLSI guidelines. Firstly, H. pylori strains were cultured in Brain Heart Infusion (BHI) medium containing 10% fetal calf serum (FCS) under microaerophilic conditions (85% N 2 , 10% CO 2 , 5% O 2 , and 90% relative humidity) using a double-gas CO 2 incubator (Binder, model CB160; Tuttlingen, Germany), while other bacterial pathogens were aerobically cultured in Mueller-Hinton (MH) broth. Candida albicans and Aspergillus fumigatus strains were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, Kawasaki, Japan) containing L-glutamine and buffered with 165 mM MOPS at pH 7.0 (denoted as RPMI medium). Subsequently, a single colony was picked and continuously incubated in BHI/MH/RPMI broth to reach a logarithmic growth phase. Then, the test compounds were dissolved in DMSO and serially diluted two-fold to different concentrations on a 96-well plate. An aliquot (10 µL) of microbial suspension was added to each well, and cell concentration was adjusted to approximately 5 × 10 5 cells/mL for H. pylori, 5 × 10 4 cells/mL for other bacterial pathogens and 1 × 10 3 cells/mL for fungi. The concentration range tested for each of the compounds was 64-0.125 µg/mL, and each compound was tested in triplicate. The negative control group was treated with sterile water. Metronidazole, methicillin, vancomycin, and amphotericin B were used as the positive control for H. pylori, S. aureus, and other bacteria and fungi, respectively. After incubating H. pylori at 37 • C for 72 h and other bacteria or fungi for 24 h, the plates were examined, and the MIC was defined as the lowest concentration of the compounds with no visible growth. Growth of seven bacterial strains exposed to compounds 1 and 2 at various concentrations after 72 h for H. pylori strains and after 24 h for other bacterial strains was examined at 600 nm for optical density, and the OD 600 was recorded. Experiments were performed with three biological replicates.
Author Contributions: C.L., J.C., D.T. and J.L., investigation, formal analysis, validation, data curation, writing-original draft preparation; D.L. and T.L., investigation, formal analysis; J.T., H.B. and B.W., conceptualization, resources, supervision, writing-original draft preparation, writing-review and editing, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The authors confirm that the data supporting the reported results are available within the article and its supplementary materials.