New Sesquiterpenoids From Plant-Associated Irpex lacteus

Bacteria produce a large number of virulence factors through the quorum sensing (QS) mechanism. Inhibiting such QS system of the pathogens without disturbing their growth is a potential strategy to control multi-drug-resistant pathogens. To accomplish this, two new tremulane-type sesquiterpenoids, irpexolaceus H (1) and I (2), along with two known furan compounds, irpexlacte B (3) and C (4), were isolated from Orychophragmus violaceus (L.) OE Schulz endophytic fungus Irpex lacteus (Fr.) Fr. Their structures were elucidated by detailed spectroscopic data (NMR, HRESIMS, IR, and UV), single-crystal X-ray diffraction, and electronic circular dichroism (ECD) analysis. Furthermore, those compounds were evaluated for anti-quorum sensing (anti-QS) activity, and compound 3 was found contributing to the potential QS inhibitory activity.


INTRODUCTION
Bacterial quorum sensing (QS) is a cell-density-dependent communication process by which cells measure population density and trigger appropriate responses and conduct behavioral regulation, such as luminescence, motility, secretion of virulence factors, and formation of biofilms (Papenfort and Bassler, 2016). QS inhibitor (QSI) inhibits the QS system without affecting bacteria's growth and reduces its virulence production and biofilm formation; thus, the bacteria are in a low or non-toxicity state, the growth is not inhibited, and therefore it is difficult to cause drug resistance (Jiang and Li, 2013). However, the purpose of traditional antibacterial agents is to kill or inhibit the growth of bacteria, and it is difficult to avoid bacterial resistance (Kalia, 2013). Therefore, finding new QSIs to replace traditional antibacterial agents has become a new strategy in the antibacterial field.
Irpex lacteus (Fr.) Fr. (Phanerochaetaceae) is a basidiomycete that usually colonizes on the deadwood white (i.e., white rot), and it is often used as a traditional Chinese medicine for the treatment of chronic glomerulonephritis. In our previous study on this subject, seven sesquiterpenoids, irpexolaceus A-G, and two new furan derivatives, irpexonjust A-B, were isolated from I. lacteus OV38 (Luo et al., 2022). Furthermore, two new tremulane-type sesquiterpenoids, irpexolaceus H (1) and I (2), were isolated in this study from fungi, as well as two furan compounds, such as irpexlacte B (3) and C (4), were also obtained ( Figure 1). These compounds were screened for QS inhibitory activity, and compound 3 exhibited the highest QS inhibitory activity among them. The details of the isolation, structure assignment, and QS inhibitory activities of 1-4 are presented.

General Experimental Procedures
Nuclear magnetic resonance (NMR) was obtained on a Bruker AV-400 spectrometer (Bruker Corporation, Karlsruhe, Germany). HRMSESI data were recorded on a Q-Exactive Orbitrap MS system (Thermo Fisher Scientific, Bremen, Germany). UV data were obtained on an Evolution 220 UVvis spectrophotometer (Thermo Fisher Scientific, Madison, United States). Infrared spectroscopy (IR) spectra were obtained on a Nicolet ™ iS10 FTIR spectrometer (Thermo Fisher Scientific, Madison, United States). Optical rotations were recorded on an Autopol VI automatic polarimeter (PerkinElmer, Waltham, MA, United States). The silica gel Qingdao Marine Chemical Factory,Qingdao,China) and Sephadex LH-20 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for open column chromatography (CC). Fractions were monitored by TLC (HSGF 254,Yantai Jiangyou Silica Gel Development Co.,Yantai,China), and spots were visualized by heating silica gel plates after soaking in methanol supplemented with 10% H 2 SO 4 . The preparative HPLC was performed with UltiMate 300 HPLC (Thermo Fisher Scientific, Madison, United States) equipped with a YMC-Pack ODS-A column (250 × 10 mmI.D, S-5 μm, 12 nm, and flow speed = 2-3 ml/min, YMC Co., Ltd., Kyoto, Japan).
In addition to the aforementioned compounds, two furans (3 and 4) were obtained and identified as irpexlacte B and C according to the previously reported data (Luo et al., 2022).

Proposed Biotransformation Pathway
Tremulane (i) and (ii) were biosynthesized based on the relative structures of 1 and 2 by the cyclization and rearrangement of farnesyl pyrophosphate (FPP) (Ayer and Browne, 1981;Ayer and Cruz, 1993). After a multi-step reaction such as esterification, cyclization, and dehydration in microbes, tremulane (i) was possibly converted to irpexlaceus H ( Figure 5). Moreover, lactarane skeletons ii-1 and ii-2 were produced by a series of methyl migrations of tremulane (ii)) (Ayer and Cruz, 1993), which differed from tremulane (i)) in the configuration of C-1 and C-7. Lactarane skeleton ii-2 was Frontiers in Chemistry | www.frontiersin.org May 2022 | Volume 10 | Article 905108 transformed to 5,6-secotremulane under the 5,6-cleavage (He et al., 2020) and then possibly converted to irpexlaceus I by a series of oxidation and dehydration ( Figure 5).

Biological Activity
Compounds 1-4 were evaluated for their QS inhibitory activities at 50 mg/ml against biomarker strains C. violaceum CV026 ( Figure 6A) and A. tumefaciens A136 ( Figure 6B). We assessed the inhibitory effect of the compound by assaying the inhibition of C6-HSL-induced violacein production by C. violaceum CV026, and C10-HSL-induced β-galactosidase expression (blue pigment) by A. tumefaciens A136. Compounds 1-2 showed no inhibitory activities against both QS systems of biomarker strains. However, compound 3, in which the binding position of hydroxyl was closer to the furan ring than 4, exhibited stronger inhibition activity against the production of violacein in C. violaceum CV026 than that of the latter but weaker inhibitory activity against the production of blue pigment in A. tumefaciens A136. The results demonstrated that the binding position of hydroxyl was vital for QS inhibitory activity.

CONCLUSION
Two new tremulane-type sesquiterpenoids, irpexolaceus H (1) and I (2), were isolated from the liquid fermentation of I. lacteus. Their structures were established based on NMR, HRESIMS, IR, singlecrystal X-ray diffraction, and ECD analysis. These compounds (1-4) were evaluated for QS inhibitory activities against C. violaceum CV026 and A. tumefaciens A136 at 50 mg/ml. The results found that compound 3 exhibited a significant QS inhibitory activity against C. violaceum CV026, and compound 4 showed a weaker activity. In addition, compound 3 also showed a weak QS inhibitory activity against A. tumefaciens A136. But interestingly, the hydroxyl binding to α-C in the furan ring showed a stronger QS inhibitory activity than that of 4 (hydroxyl binding to β-C in the furan ring), which suggested that the position of hydroxyl in the furan ring was possibly vital for QS inhibitory activity against C. violaceum CV026.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. These data can be found at: https://www.ccdc.cam.ac. uk/, 2133065, 2095205, 2095201, and 2095200.

AUTHOR CONTRIBUTIONS
H-ZL contributed to the chemical and biological experiments and prepared the manuscript draft. HJ contributed to the spectra data analysis. X-SH contributed to the ECD calculation and analysis. Frontiers in Chemistry | www.frontiersin.org May 2022 | Volume 10 | Article 905108