New Polyene Macrolide Compounds from Mangrove-Derived Strain Streptomyces hiroshimensis GXIMD 06359: Isolation, Antifungal Activity, and Mechanism against Talaromyces marneffei

Mangrove-derived actinomycetes represent a rich source of novel bioactive natural products in drug discovery. In this study, four new polyene macrolide antibiotics antifungalmycin B-E (1–4), along with seven known analogs (5–11), were isolated from the fermentation broth of the mangrove strain Streptomyces hiroshimensis GXIMD 06359. All compounds from this strain were purified using semi-preparative HPLC and Sephadex LH-20 gel filtration while following an antifungal activity-guided fractionation. Their structures were elucidated through spectroscopic techniques including UV, HR-ESI-MS, and NMR. These compounds exhibited broad-spectrum antifungal activity against Talaromyces marneffei with minimum inhibitory concentration (MIC) values being in the range of 2–128 μg/mL except compound 2. This is the first report of polyene derivatives produced by S. hiroshimensis as bioactive compounds against T. marneffei. In vitro studies showed that compound 1 exerted a significantly stronger antifungal activity against T. marneffei than other new compounds, and the antifungal mechanism of compound 1 may be related to the disrupted cell membrane, which causes mitochondrial dysfunction, resulting in leakage of intracellular biological components, and subsequently, cell death. Taken together, this study provides a basis for compound 1 preventing and controlling talaromycosis.

Mangroves are ecologically significant plants in marine habitats that inhabit the coastlines of many countries [17].Being a highly productive and diverse ecosystem, mangroves are rich in numerous classes of actinomycete that are of great importance in the field of antibiotics [18].Furthermore, about 200 compounds, such as salinosporamide A (to be processed for clinical trials for cancer treatment), xiamycins, rifamycins, and antimycin A were discovered from mangrove actinobacteria, which have become an important source of novel bioactive compounds [19][20][21][22][23].
In our preliminary study, Streptomyces hiroshimensis GXIMD 06359 was isolated from mangrove in the west coast of Hainan [24], which was identified as the potential strain to produce antimicrobial active metabolites against T. marneffei in Am2ab medium.The scaledup fermentation and extensive chromatographic separation of the EtOAc extract resulted in the isolation of four new metabolites, namely antifungalmycins B-E (1-4), together with seven known compounds (5)(6)(7)(8)(9)(10)(11). Herein, we report the isolation and structural determination of these compounds (Figure 1) along with the antifungal activities.Then the mechanism of compound 1 inhibiting T. marneffei was studied, with the aim of finding potential new drugs for the precision treatment of talaromycosis.
Mar. Drugs 2024, 22, x FOR PEER REVIEW 2 mangroves are rich in numerous classes of actinomycete that are of great importan the field of antibiotics [18].Furthermore, about 200 compounds, such as salinosporam A (to be processed for clinical trials for cancer treatment), xiamycins, rifamycins, and timycin A were discovered from mangrove actinobacteria, which have become an portant source of novel bioactive compounds [19][20][21][22][23].
In our preliminary study, Streptomyces hiroshimensis GXIMD 06359 was isolated mangrove in the west coast of Hainan [24], which was identified as the potential stra produce antimicrobial active metabolites against T. marneffei in Am2ab medium.scaled-up fermentation and extensive chromatographic separation of the EtOAc ex resulted in the isolation of four new metabolites, namely antifungalmycins B-E (1-4 gether with seven known compounds (5)(6)(7)(8)(9)(10)(11). Herein, we report the isolation and s tural determination of these compounds (Figure 1) along with the antifungal activ Then the mechanism of compound 1 inhibiting T. marneffei was studied, with the ai finding potential new drugs for the precision treatment of talaromycosis.

Structural Elucidation
Compound 1 was obtained as a light-yellow amorphous powder.Its HR-ESI-MS spectrum showed a characteristic [M − H] − ion peak at m/z 675.3578   Careful analysis of the 1 H and 13 C NMR data (Table 1) of 1 showed they were very similar to antifungalmycin [25,26], which possesses the same lactone ring.The main difference was that the signals for two methylene groups [(δ H 1.24, δ C 31.3, CH 2 -4 ′ ) and (δ H 1.25, δ C 22.1, CH 2 -5 ′ )] at the side chain in the NMR spectra of antifungalmycin were absent in compound 1.Those were supported by the 1 H-1 H COSY and HMBC correlations (Figure 2).In the 1 H-1 H COSY spectrum of compound 1, correlations were observed for Thus, the planar structure of 1 was determined.In the NOESY spectrum, the correlations of H-3/H-1 ′ , H-13/H-15, H-25/H-26 (Figure 3) were not enough to elucidate the compound 1 relative configuration.Consequently, compound 1's relative configuration has not been elucidated, and is named antifungalmycin B.
Compound 2, isolated as a light-yellow amorphous powder.The molecular formula was deduced to be C 33 H 56 O 11 by the HR-ESI-MS peak at m/z 627.3743 [M−H] − (calcd for 627.3744), indicating 6 degrees of unsaturation.The 1 H NMR spectrum (Figure S10) shows resonances for three methyl protons δ H 1.19 (3H, d, J = 6.3 Hz, H 3 -26), 0.84 (3H, m, H 3 -27), and 0.84 (3H, t, J = 6.9 Hz, H 3 -6 ′ ); eight methylene protons   Thus, the planar structure of 1 was determined.In the NOESY spectrum, the correlations of H-3/H-1′, H-13/H-15, H-25/H-26 (Figure 3) were not enough to elucidate the compound 1 relative configuration.Consequently, compound 1's relative configuration has not been elucidated, and is named antifungalmycin B.   Compound 2, isolated as a light-yellow amorphous powder.The molecular formula was deduced to be C33H56O11 by the HR-ESI-MS peak at m/z 627.3743 [M−H] − (calcd for 627.3744), indicating 6 degrees of unsaturation.The 1 H NMR spectrum (Figure S10) shows resonances for three methyl protons δH 1.19 (3H, d, J = 6.3 Hz, H3-26), 0.84 (3H, m, H3-27), and 0.84 (3H, t, J = 6.9 Hz, H3-6′); eight methylene protons δH 1.92 (1H, m, H-12a), 1. 52   The 1 H and 13 C NMR data (Table 1) of 2 suggested that it was similar to antifungalmycin [25,26].Combined with analyzing the 1 H-1 H COSY and HMBC spectra, it was revealed that compound 2 was a 26-membered macrocyclic lactone ring.Compared with antifungalmycin, the signals for one-quarter carbon (δC 82.0) and one methylene were absent in the lactone ring in compound 2. In the COSY spectrum, correlations were observed for H- The 1 H and 13 C NMR data (Table 1) of 2 suggested that it was similar to antifungalmycin [25,26].Combined with analyzing the 1 H-1 H COSY and HMBC spectra, it was revealed that compound 2 was a 26-membered macrocyclic lactone ring.Compared with antifungalmycin, the signals for one-quarter carbon (δ C 82.0) and one methylene were absent in the lactone ring in compound 2. In the COSY spectrum, correlations were observed for The NMR data (Table 1) of 3 suggested that it was similar to antifungalmycin [25,26].Detailed analysis of the 1 H-1 H COSY and HMBC spectra revealed that the main differences between them were the positions of conjugated double bonds.The conjugated double bonds of 3 were in C-18 to C-25, but in antifungalmycin were in C-17 to C-24.In the COSY spectrum, correlations were observed for H  2). Thus, the planar structure of 3 was determined.In the NOESY spectrum, the correlations of H-3/H-1 ′ , H-13/H-14/H-15 (Figure 3) were not enough to elucidate the compound 3 relative configuration.Consequently, compound 3's relative configuration has not been elucidated, and is named antifungalmycin D.
The known compound 11 was obtained as a light-yellow amorphous powder.It was identified as filipin I [29], by comparing 1 H-NMR, 13 C-NMR, and HR-ESI-MS data with that reported.

Antifungal Activity of the Compounds
The antifungal potency of the compounds against T. marneffei was evaluated by MIC and MFC values; the results are shown in Table 2.In our experiment, most of the compounds exhibited antifungal activity except compound 2, which has an MIC value of more than 128 µg/mL.In addition, compound 9 exhibited the best antifungal activity against T. marneffei, with MIC and MFC values of 2 and 4 µg/mL, respectively.Moreover, compound 1 showed the lowest MIC and MBC values, which represented the best antifungal activity of all the new compounds.So, we further explore the antifungal mechanism of compound 1 in the next experiment.

Compound 1 Inhibited the Growth of T. marneffei
To further analyze the growth-inhibiting characteristics of compound 1, the time course of T. marneffei growth in the presence of 1 at different concentrations was plotted.T. marneffei exhibited rapid growth in the control group, and the logarithmic growth stage was achieved within 40 h of incubation, then entered a stabilization phase after 60 h of incubation (Figure 4A).However, the growth of T. marneffei after treatment with 1 at 1/2 MIC and 1 MIC showed a substantially lower growth rate than that of the control.Moreover, before 60 h of incubation, no further growth of T. marneffei was observed in 1/2 MIC and 1 MIC compound 1-treated groups.But after 60 h of incubation, T. marneffei with 1 continued to grow, though at a lower rate than control, and the fungi growth rate of group compound 1 at 1 MIC was lower than group compound 1 at 1/2 MIC.Overall, these results confirmed that 1 had an inhibitory effect on T. marneffei growth, and was shown to be concentration-dependent and time-limited.

Compound 1 Disrupted the Cell Membrane of T. marneffei
The membrane integrity of the fungi was investigated to verify the ability of compound 1 to damage the fungal cell membrane.The electric conductivity of the cell suspensions implied the permeability of the cell membrane.Electrolytes are charged molecules such as sodium chloride and potassium chloride, and they are essential for fungal metabolism and growth [30].Thus, their leakage can lead to fungal inhibition or death.Compared with the control, compound 1 resulted in a significant increase in conductivity.The conductivity of T. marneffei increased significantly from 1.80 for the control to 2.99 and 3.69 in the presence of compound 1 at the levels of 1/2 MIC and 1 MIC after 15 h, respectively (Figure 4B).Moreover, after 9 h exposure to the 1/2 MIC of compound 1, the extracellular conductivity entered a steady stage.However, for the group of 1 MIC, the extracellular conductivity continued to increase.This indicates that compound 1 has a destructive effect on the cell membrane of T. marneffei, and shows a concentration dependence consistent with the growth-inhibiting results.
In order to further determine the degree of cell membrane damage by compound 1, in the current work, nucleic acids and proteins released from the cytoplasm were monitored by the detection of absorbance at 260 nm and 280 nm, respectively.As the previous work reported, nucleic acid and protein play important roles in bacterial metabolism as they dominate the genetic information and cellular structure [31].Leakage of cellular materials was analyzed by detecting 260 nm and 280 nm absorbing materials.Therefore, the absorbance of the material and proteins at 260 nm and 280 nm wavelengths can be used as an indicator of damage to the cell wall and membrane, which causes leakage of the cellular materials into the surroundings [32].As shown in Figure 4C,D, both cell constituents were released rapidly from T. marneffei into cell suspensions and their amounts increased multi-fold after treatment with compound 1.In addition, there was a progressive release of proteins and nucleic acids from T. marneffei after exposure to compound 1 for 4 h, followed by a steady state.Moreover, the leakage of nucleic acids and proteins in the group treated with 1 MIC compound 1 was larger than the control and 1/2 MIC compound 1 group.Compound 1 dose-dependently destroyed the cell membrane of T. marneffei, which was consistent with the previous results.Similar results have also been reported for the crude methanolic extract of Myrtus communis roots and leaves when tested against Candida glabrata, showing increased absorbance at a wavelength of 260 nm [33].In this study, compound 1 was efficacious in inhibiting or killing the fungi by damaging their cell membranes, resulting in the leakage of the 260 nm and 280 nm absorbing materials, such as DNA, RNA, and proteins, which are essential for fungal growth.
To further investigate the mechanisms underlying compound 1's disruption of the cell membrane in T. marneffei cells, the Na + /K + -ATPase and Ca 2+ -ATPase activities of T. marneffei cells were detected.Na + /K + -ATPase is a carrier protein that exists in the phospholipid bilayer of cells.It mainly controls the transmembrane transport of Na + and K + .It can release energy by decomposing ATP, and uses this energy to transport Na + and K + [34].Ca 2+ -ATPase is a membrane transport protein ubiquitously found in the endoplasmic reticulum of all eukaryotic cells.As a calcium transporter, Ca 2+ -ATPase maintains a low cytosolic calcium level that enables a vast array of signaling pathways and physiological processes [35].Na + /K + -ATPase and Ca 2+ -ATPase are important components of cell membrane transport.The Na+/K + -ATPase and Ca 2+ -ATPase activities of T. marneffei cells are shown in Figure 4E,F.Compared with the control group without 1, the Na + /K + -ATPase and Ca 2+ -ATPase activities of the experimental group with compound 1 were significantly decreased.Among them, the experimental group with 1 at the levels of 1 MIC had the largest decrease.This showed that compound 1 had a certain inhibitory effect on the Na + /K + -ATPase and Ca 2+ -ATPase activities of T. marneffei, which is consistent with the above results.
These results indicated that the antibacterial action mode of compound 1 against T. marneffei probably involved the alteration of the structure of cell wall and membrane, causing the loss of cell viability.

Effect of Compound 1 on Morphology of T. marneffe
The morphological and ultrastructural changes in T. marneffei treated with compound 1 for 72 h were observed by SEM and TEM to better understand the antifungal mode of action of compound 1.For T. marneffei cells, deformation was the most significant feature, which was apparent in the SEM image (Figure 5).The T. marneffei cells treated with compound 1 at 1/2 MIC displayed distorted membrane morphology, disruption of cell membrane, and leakage of cellular contents; and those treated with 1 MIC displayed distorted membrane morphology.Furthermore, a proportion of T. marneffei cells treated with compound 1 showed abnormalities in the TEM images, including the disappearance of cell wall, disruption of cell membrane, thinning of cytoplasm, distortion of cells, heterogeneous distribution of melanin, and leakage of intracellular materials (Figure 4G).S.K.P. Lau et al. reported that T. marneffei in yeast form can cause infections, and produce melanin as well, which plays an important role in the pathogenicity of T. marneffei [36].Therefore, the decrease in intracellular melanin may also be the pathway of compound 1 inhibiting T. marneffei.These findings supported the results of the leakage of extracellular conductivity, nucleic acids and proteins leakage analysis, and Na + /K + -ATPase and Ca 2+ -ATPase activities in the present study.
Na + /K + -ATPase and Ca 2+ -ATPase activities of T. marneffei, which is consistent with above results.
These results indicated that the antibacterial action mode of compound 1 again marneffei probably involved the alteration of the structure of cell wall and membrane, c ing the loss of cell viability.

Effect of Compound 1 on Morphology of T. marneffe
The morphological and ultrastructural changes in T. marneffei treated with pound 1 for 72 h were observed by SEM and TEM to better understand the antifu mode of action of compound 1.For T. marneffei cells, deformation was the most signifi feature, which was apparent in the SEM image (Figure 5).The T. marneffei cells tre with compound 1 at 1/2 MIC displayed distorted membrane morphology, disruptio cell membrane, and leakage of cellular contents; and those treated with 1 MIC displ distorted membrane morphology.Furthermore, a proportion of T. marneffei cells tre with compound 1 showed abnormalities in the TEM images, including the disappear of cell wall, disruption of cell membrane, thinning of cytoplasm, distortion of cells, h ogeneous distribution of melanin, and leakage of intracellular materials (Figure S.K.P. Lau et al. reported that T. marneffei in yeast form can cause infections, and pro melanin as well, which plays an important role in the pathogenicity of T. marneffei Therefore, the decrease in intracellular melanin may also be the pathway of compou inhibiting T. marneffei.These findings supported the results of the leakage of extracel conductivity, nucleic acids and proteins leakage analysis, and Na + /K + -ATPase and ATPase activities in the present study.

Effects of Compound 1 on Mitochondrial Function
Mitochondria are key energy and metabolic regulatory centers within cells and also play an important role in maintaining cell growth and survival in mycelial cells.The core function of mitochondria is to synthesize ATP through oxidative phosphorylation.Therefore, the normal conduct of mitochondrial oxidative phosphorylation and the TCA cycle, especially the activities of related enzymes, is essential for maintaining cell survival.ATPase has an important role in energy metabolism [37].The results of ATPase content in T. marneffei cells are shown in Figure 6A.The ATP content of the control group was 23,693.5 µmol/gprot.After treatment with 1 MIC compound 1, the intracellular ATP level reduced to 8151.1 µmol/gprot, which was a 65.6% reduction (p < 0.05).In addition, after treatment with 1/2 MIC compound 1, the intracellular ATP level reduced to 16,213.2µmol/gprot, which was a 31.6%reduction (p < 0.05).ATPase content was significantly decreased after compound 1 treatment.These results indicated that the antifungal activity of compound 1 against T. marneffei can be attributed to disruption of the respiratory chain.
synthesis, TCA cycle, and C4 cycle.Compared with the control, compound 1 resulted in a reduction in the activity of SDH and MDH.The SDH activity of compound 1 at the levels of 1/2 MIC and 1 MIC was significantly reduced compared with the control (p < 0.05) (Figure 6B), and the MDH activity of compound 1 at the levels of 1/2 MIC and 1 MIC was considerably lower than that of the control (p < 0.05) (Figure 6C).The above experimental results showed that MDH and SDH activities decreased with increasing compound 1 concentration in T. marneffei (Figure 6A,B), which suggested that compound 1 disrupts mitochondrial function by affecting MDH and SDH activities.
The above results suggested that compound 1 blocked the respiratory chain and energy metabolism, thereby killing the fungi.

Discussion
Polyene macrolide antibiotics are a significant group of antibiotics and have an important role in the treatment of fungal infections [39].For example, amphotericin B, pimamycin, and nystatin have been widely used in clinical treatment [40].Many researchers suggest that most polyene macrolides are bioactive compounds with a wide range of antifungal activity.However, the high hemolytic toxicity, poor water solubility, and unstable exposure to light limit the development of some compounds with good antifungal activity into clinical drugs.For a long time, researchers have been committed to chemical SDH (Succinate dehydrogenase) is a part of the respiratory chain (complex II).SDH catalyzes the oxidation of succinate to fumaric acid and FADH2.Therefore, it connects the TCA cycle with the respiratory chain, and the generated FADH2 does not dissociate from the enzyme, which directly uses the electrons to reduce the coenzyme Q, and then passes it to the complex III [38].MDH (Malate dehydrogenase) can catalyze the reversible conversion between malic acid and oxaloacetate, and is also an important enzyme in mitochondrial function, which is mainly involved in some metabolic pathways such as photosynthesis, TCA cycle, and C4 cycle.Compared with the control, compound 1 resulted in a reduction in the activity of SDH and MDH.The SDH activity of compound 1 at the levels of 1/2 MIC and 1 MIC was significantly reduced compared with the control (p < 0.05) (Figure 6B), and the MDH activity of compound 1 at the levels of 1/2 MIC and 1 MIC was considerably lower than that of the control (p < 0.05) (Figure 6C).The above experimental results showed that MDH and SDH activities decreased with increasing compound 1 concentration in T. marneffei (Figure 6A,B), which suggested that compound 1 disrupts mitochondrial function by affecting MDH and SDH activities.
The above results suggested that compound 1 blocked the respiratory chain and energy metabolism, thereby killing the fungi.

Discussion
Polyene macrolide antibiotics are a significant group of antibiotics and have an important role in the treatment of fungal infections [39].For example, amphotericin B, pimamycin, and nystatin have been widely used in clinical treatment [40].Many researchers suggest that most polyene macrolides are bioactive compounds with a wide range of antifungal activity.However, the high hemolytic toxicity, poor water solubility, and unstable exposure to light limit the development of some compounds with good antifungal activity into clinical drugs.For a long time, researchers have been committed to chemical derivation, structural modification, genetic engineering, combined biosynthesis, and other methods to improve the antibacterial activity and solubility of these compounds and reduce the hemolytic toxicity.This has made certain research progress, but far from enough.Extensive research has shown that some bioactive secondary metabolites of marine microbial origin with strong antibacterial and antifungal activities are being intensely used as antibiotics and may be effective against infectious diseases [41].In our study, four new compoundstetrene macrolide compounds (1)(2)(3)(4), and seven known polyene macrolide antibiotics, were isolated from the fermentation broth of the mangrove strain S. hiroshimensis GXIMD 06359.Their structures, including their relative configurations, were determined by HR-ESI-MS and NMR spectra.The antifungal activity of the compounds against T. marneffei was measured by detection of MIC and MBC in the present study.The results demonstrated that all compounds except compound 2 exhibit antifungal and fungicidal properties against T. marneffei.Moreover, the lowest MIC and MFC of the new compounds was compound 1.To further analyze the antifungal activity of 1, the time course of T. marneffei growth in the presence of 1 at different concentrations was plotted.These results demonstrated that 1 can significantly inhibit the growth of T. marneffei.Therefore, compound 1 could be considered an effective antibacterial agent; we further explore the mechanism of compound 1 inhibiting T. marneffei.
Cell membranes have important physiological functions, including maintaining the stability of the intracellular environment, signal transduction, and material transportation [42].The integrity of cell membranes is crucial for cell viability, and membrane damage can lead to high cytotoxicity.Numerous studies have shown that the cell membrane of fungi is a target for inhibiting fungal growth and reproduction [43].The previous studies have reported that the antifungal mechanism of action of polyene macrolides is binding to the fungal surface, which produces membrane breakdown, resulting in leakage of protein and vital nutrients and, ultimately, cell death [44][45][46].In this study, it was found that compound 1 caused significant damage to cell membranes.Compound 1 irreversibly damaged the plasma membrane of T. marneffei cells.Its treatment increased extracellular conductivity, proteins, and nucleic acids in T. marneffei cultures, suggesting electrolyte leakage due to reduced membrane integrity of T. marneffei cells.Moreover, the decrease in Na + /K + -ATPase and Ca 2+ -ATPase activities confirmed the destruction of cell membrane function.SEM and TEM results confirmed that compound 1 treatment disrupted the integrity of T. marneffei cell walls and membranes.Cell wall and membrane integrity are critical for maintaining fungal viability.Kamble, M. T. et al. reported that SGF disrupted the bacterial cell membrane, resulting in leakage of intracellular biological components, and subsequently, cell death, in Vibrio parahaemolyticus and Vibrio harveyi, which is similar to our results [47].
In addition to destructing the cell membrane, mitochondrial dysfunction plays an important role in the potential mechanisms of antifungal drugs [48].Xin et al. reported that antofine against P. digitatum is related to the cell membrane integrity and energy metabolism by affecting intracellular ATP content [49].Pristimerin has been reported to exert antifungal activity; it caused mitochondrial membrane damage and affected mitochondria structure and functions, then oxidative phosphorylation and TCA cycle were inhibited, and energy metabolism was blocked in S. sclerotiorum [50].In this study, we also investigated the role of mitochondrial function pathways against T. marneffei.Our results indicated that compound 1 caused a significant decrease in intracellular ATP levels and a significant decrease in the activities of MDH and SDH, and this was shown to be concentration-dependent.These results indicated that the antifungal activity of compound 1 against T. marneffei can be attributed to disruption of the respiratory chain.Therefore, the death of T. marneffei may be caused by mitochondrial dysfunction, in turn caused by the destruction of cytoplasmic membrane permeability and integrity.
This study showed that compound 1 effectively prevented T. marneffei growth.Compound 1 disrupts cytoplasmic membrane permeability and integrity, causes mitochondrial dysfunction, and T. marneffei metabolic disorders.We speculate that the antifungal mechanism of compound 1 on T. marneffei is through the destruction of T. marneffei cell membrane integrity and mitochondrial function to induce apoptosis.Compound 1 showed promising potential as a drug against T. marneffei.But the detailed role of compound 1 in bacterial membranes is unclear and needs further investigation.

Actinomycete Material
The strain S. hiroshimensis GXIMD 06359 was isolated from mangrove in the west coast of Hainan [24].This strain is stored at Institute of Marine Drugs, Guangxi University of Chinese Medicine.

Fermentation, Extraction, and Isolation
After activation, S. hiroshimensis GXIMD 06359 was inoculated into 1 L flapper conical flask (containing 300 mL Am2ab medium, sterilized) and fermented in a constant temperature shaking table at 28 • C and 180 r/min for 10 days.After fermentation, the fermentation solution was filtered, and the bacterial solution and bacteria were separated.The bacterial solution was extracted with equal volume ethyl acetate three times, and the bacteria were soaked in equal volume acetone and extracted by ultrasound for 20 min until nearly colorless.The ethyl acetate phase and acetone phase fermentation crude extracts were obtained after concentration under reduced pressure.The crude extract (200.2 g) was subjected to normal phase silica gel column chromatography and gradient elution was performed with chloroform/acetone system (10:0, 10:2, 10:4, 10:8) and chloroform/methanol system (10:1, 10:2, 10:4, 0:10).The collected fractions were analyzed by thin layer chromatography (TLC) and HPLC.A total of 13 fractions (Fr.A1-A13) were obtained.

Microbial Strains OriginA and Culture Conditions
Reference strains (Talaromyces marneffei ATCC) were from YE Li from Guangxi Medical University (Guangxi Key Laboratory of AIDS Prevention and Control, School of Public Health, Guangxi Medical University).Seven-day-old pure culture of the yeast form grown on brain-heart infusion (BHI) agar was used in all reactions.The colonies of T. marneffei were flooded with Phosphate buffer saline (PBS) and the number of fungi was counted with a hemocytometer after washing three times.The cells were suspended in PBS and thoroughly vortexed.The suspensions were added to RPMI 1640 medium to obtain a stock of 1-5 × 10 6 CFU/mL that was then diluted 1:100, resulting in a working stock of 1-5 × 10 4 CFU/mL.

Antifungal Activity
Antifungal susceptibility testing was performed using the microdilution method according to CLSI protocol M27-A3 (Clinical and Laboratory Standards Institute) with minor modifications [51].Candida parpsilosis ATCC 22019 was included as quality control through for all experiments.Wells containing inoculum alone and inoculum with DMSO were used as negative controls.AMB and FLC were used as a positive control.The minimum inhibitory concentration (MIC) was defined as the lowest concentration resulting in 100% inhibition of visible fungal growth after incubation at 37 • C for 72 h.

Determination of Minimal Fungicidal Concentration (MFC)
The MFCs of compounds were determined according to the methods of Mbah et al. [52].Briefly, 10 µL from wells corresponding to 1, 2, 3, and 4-fold of the MIC, were placed on a Sabouraud Dextrose Agar (SDA) and incubated at optimal temperatures for 72 h.MFC was defined as the lowest concentration with no fungal growth.Exponentially growing yeast cells were harvested and resuspended in RPMI-1640 to obtain a final concentration of 1-5 × 10 4 CFU/mL.Different concentrations of compound 1 were added to the cells.Cells were incubated under shaking 180 rpm at 37 • C, and 10 µL from suspensions were placed on SDA and incubated at optimal temperatures for 72 h, then measured at the indicated time points after incubation (0, 12, 24, 36, 48, 60, and 72 h).The same volumes of solvents (DMSO) were added to the untreated controls.Three independent experiments were performed for optimal results.

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)
SEM was used to observe the morphological changes of compound 1-treated T. marneffei.The fungal cells obtained from the logarithmic growth phase were treated with the compound 1 at 1/2 and 1-fold of the MIC value at 37 • C for 72 h.Then, the suspensions were centrifuged at 12,000 rpm/min for 10 min.The sediments were washed with 0.1 M PBS, (pH = 7.2) and fixed with 2.5% glutaraldehyde in PBS for 2 h at 4 • C. The cells were washed in the same buffer and were post-fixed for 30 min with osmium tetroxide.After harvesting, the cells were further dehydrated via graded ethanol concentrations (30%, 50%, 70%, 90%, and 100%) for 10 min each.Untreated cells were similarly processed and used as control.Then, cells were fixed on SEM support and observed by SEM (Sigma300, Zeiss), Wuhan, Hubei, China.
The pretreatment of fungal cells for transmission electron microscopy (TEM) were the same as that for scanning electron microscopy (SEM, Wuhan, Hubei, China).After being fixed with 2.5% glutaraldehyde, post-fixed by 1% osmic acid, dehydrated using alcohol, permeated using white resin, and embedded by roasting at 55 • C, the samples were cut into thin sections to perform TEM (HITACHI HT 7800 120 kv, Wuhan, Hubei, China).

Leakage of Extracellular Conductivity
Fungal membrane permeability was determined and expressed as the electric conductivity according to the method by Maliehe, T. S. et al. [53].Fungal cells were cultivated at 37 • C to mid-exponential stage and collected by centrifugation (8000 rpm for 15 min).Cells were washed twice in 0.1 M PBS.The different concentrations compound 1 were added into the isotonic fungal suspensions (1 × 10 4 CFU/mL) and incubated at 37 • C for 15 h.Thereafter, their conductivities were measured and recorded as A 1 (0, 3, 6, 9, 12, 15 h).The conductivities of the fungi in 0.1 M PBS treated with boiling water for 5 min were used as the control and marked as A 0 .The cell membrane permeability was then calculated using the formula: Electric conductivity = A 1 − A 0 .4.7.4.Leakage of 260 nm and 280 nm Absorbing Material Fungal strains were cultured in RPMI-1640 and incubated at 37 • C for 12 h.The most active compound 1 were added to the fungal suspensions at 1-fold and 1/2-fold of the MIC values.Suspensions were incubated at 37 • C and samples were removed at times 0, 2,

Figure 4 .
Figure 4. (A) Time-kill curve of compound 1 against T. marneffei.(B) The effect of compound 1 on the extracellular conductivity of T. marneffei.(C) The effect of compound 1 on the extracellular nucleic acids of T. marneffei.(D) The effect of compound 1 on the extracellular proteins of T. marneffei.(E) The effect of compound 1 on Na +/ K + -ATPase activity against T. marneffei.(F) The effect of compound 1 on Ca 2+ -ATPase activity against T. marneffei.(G) TEM of T. marneffei treated with compound 1 for 72 h.Data are shown as mean ± S.D. * p < 0.05, ** p < 0.01, and *** p < 0.001; Student's t test, n = 3.

Figure 5 .
Figure 5. SEM of T. marneffei treated with compound 1 for 72 h.Figure 5. SEM of T. marneffei treated with compound 1 for 72 h.

Figure 5 .
Figure 5. SEM of T. marneffei treated with compound 1 for 72 h.Figure 5. SEM of T. marneffei treated with compound 1 for 72 h.

Table 2 .
MIC and MFC of the compounds.