Activity of the pure compounds (2E)–cillifuranone, taiwapyrone, and pachybasin isolated from the fungus Neodidymelliopsis sp. against Mycobacterium abscessus and M. marinum.

The ascomycete fungus Neodidymelliopsis sp. (ICMP 11463) was isolated in October 1991 from a leaf spot on New Zealand native Pittosporum. Fifty-five Potato Dextrose Agar plates were inoculated with ICMP 11463 and incubated at room temperature for 4 weeks. Fully grown fungal plates were freeze-dried (108.6 g, dry weight) and extracted with MeOH (2 × 500 mL) for 4 h followed CH2Cl2 (2 × 500 mL) overnight. Combined organic extracts were concentrated under reduced pressure to afford a red/brown gum (0.51 g). The crude product was subjected to C8 reversed-phase column chromatography eluting with a gradient of H2O/MeOH to afford five fractions (F1–F5). The pure compounds taiwapyrone, pachybasin, and (2E)–cillifuranone were obtained after further fractionation by Sephadex LH-20 and silica gel column chromatography. Antimicrobial evaluation of the pure compounds was assessed against Mycobacterium abscessus and M. marinum. Because of the slow growth of many mycobacterial species, we routinely use luciferase-tagged strains for our assays. M. abscessus BSG301 and M. marinum BSG101 (1) are stable bioluminescent derivatives transformed with the integrating plasmid pMV306G13ABCDE (2). As bacteria only produce light when alive, bioluminescence is an excellent non-destructive real-time reporter to assay for anti-mycobacterial activity in microtitre plate formats using a luminometer (1,3,4) or in vivo using sensitive imaging equipment (5). Mycobacterial cultures were grown with shaking (200 rpm) in Middlebrook 7H9 broth (Fort Richard, Auckland) supplemented with 10% Middlebrook ADC enrichment media (Fort Richard), 0.4% glycerol (Sigma-Aldrich) and 0.05% tyloxapol (Sigma-Aldrich). M. abscessus was grown at 37 °C and M. marinum at 28 °C. Cultures were grown until they reached stationary phase (approximately 3-5 days for M. abscessus BSG301 and 7-10 days for M. marinum BSG101) and then diluted in Mueller Hinton broth II (MHB) (Fort Richard) supplemented with 10% Middlebrook ADC enrichment media and 0.05% tyloxapol to give an optical density at 600 nm (OD600) of 0.001 which is the equivalent of ~106 bacteria per mL. Pure compounds were dissolved in DMSO and added in duplicate to the wells of a black 96-well plate (Nunc, Thermo Scientific) at doubling dilutions with a maximum concentration of 128 μg/mL. Then, 50 μL of diluted bacterial culture was added to each well of the compound containing plates giving final compound concentrations of 0-64 μg/mL and a cell density of ~5 × 105 CFU/mL. Rifampicin (Sigma-Aldrich) was used as positive control at 1000 μg/mL for M. abscessus and 10 μg/mL for M. marinum. Between measurements, plates were covered, placed in a plastic box lined with damp paper towels and incubated with shaking at 100 rpm at 37 °C for M. abscessus and 28 °C for M. marinum. Bacterial luminescence (as relative light units (RLU) was measured at regular intervals using a Victor X-3 luminescence plate reader (PerkinElmer) with an integration time of 1 s. More detailed protocols are available at protocols.io (6, 7).References:1. Dalton JP, Uy B, Okuda K, Hall CJ, Denny WA, Crosier PS, Swift S, Wiles S (2017). Screening of anti-mycobacterial compounds in a naturally infected zebrafish embryo model. Journal of Antimicrobial Chemotherapy 72(2):421-427 (doi: 10.1093/jac/dkw421).2. Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible UE, Robertson BD, Wiles S (2010). Optimisation of bioluminescent reporters for use with Mycobacteria. PLOS One. 5(5): e10777 (doi:10.1371/journal.pone.0010777).3. Andreu N, Fletcher T, Krishnan N, Wiles S, Robertson BD (2012). Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence. Journal of Antimicrobial Chemotherapy. 67(2): 404-14 (doi: 10.1093/jac/dkr472). 4. Dalton JP, Uy B, Phummarin N, Copp BR, Denny WA, Crosier PS, Swift S, Wiles S (2016). Effect of common and experimental anti-tuberculosis treatments on Mycobacterium tuberculosis growing as biofilms. PeerJ. 4:e2717 (doi: 10.7717/peerj.2717).5. Andreu N, Zelmer A, Sampson SL, Ikeh M, Bancroft GJ, Schaible UE, Wiles S, Robertson BD (2013). Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase. Journal of Antimicrobial Chemotherapy. 68(9):2118-27 (doi: 10.1093/jac/dkt155).6. Grey A & Wiles S (2021). Bioluminescence-based Minimum Inhibitory Concentration (MIC) testing of pure compounds isolated from fungi against Mycobacterium marinum. Protocols.io. (doi: dx.doi.org/10.17504/protocols.io.3x7gprn). 7. Grey A & Wiles S (2021). Bioluminescence-based Minimum Inhibitory Concentration (MIC) testing of pure compounds isolated from fungi against Mycobacterium abscessus. Protocols.io. (doi: dx.doi.org/10.17504/protocols.io.bumcnu2w).

自1991年10月，从新西兰本土植物蒲桃的叶斑病中分离得到子囊菌真菌Neodidymelliopsis sp.（ICMP 11463）。该真菌被接种于55个马铃薯葡萄糖琼脂平板上，并在室温下培养4周。完全生长的真菌平板经冷冻干燥（干重108.6克）后，用甲醇（2×500毫升）提取4小时，随后用二氯甲烷（2×500毫升）过夜萃取。将合并的有机提取物在减压下浓缩，得到红色/棕色树脂（0.51克）。粗产品经过C8反相柱层析，以水/甲醇梯度洗脱，得到五个馏分（F1-F5）。通过Sephadex LH-20和硅胶柱层析进一步分离，获得了三种纯净化合物：台湾菌素、厚果菌素和（2E）-毛茛酮。这些纯净化合物对胞内分枝杆菌和M. marinum进行了抗菌活性评估。鉴于许多分枝杆菌种类的生长缓慢，我们通常使用标记有荧光素酶的菌株进行我们的检测。M. abscessus BSG301和M. marinum BSG101（1）是稳定的生物发光衍生物，经整合质粒pMV306G13ABCDE（2）转化。由于细菌仅在存活时才产生光，因此生物发光是一种优秀的非破坏性实时报告系统，可用于在微孔板格式中使用发光计（1,3,4）或使用敏感成像设备（5）进行抗分枝杆菌活性检测。分枝杆菌培养物在200 rpm的摇床中用Middlebrook 7H9肉汤（Fort Richard，奥克兰）进行培养，并补充了10%的Middlebrook ADC富集培养基（Fort Richard）、0.4%甘油（Sigma-Aldrich）和0.05%吐温80（Sigma-Aldrich）。M. abscessus在37°C下培养，而M. marinum在28°C下培养。培养物培养至稳定期（M. abscessus BSG301约为3-5天，M. marinum BSG101约为7-10天），然后稀释在含有10% Middlebrook ADC富集培养基和0.05%吐温80的Mueller Hinton broth II（MHB）（Fort Richard）中，以获得600纳米处的吸光度（OD600）为0.001，相当于每毫升约含有106个细菌。纯净化合物溶解于二甲基亚砜中，并以双倍稀释加入黑色96孔板（Nunc，Thermo Scientific）的孔中，最大浓度为128 μg/mL。然后，向含有化合物的孔中各加入50 μL稀释的细菌培养物，得到最终化合物浓度为0-64 μg/mL和细胞密度约为5 × 105 CFU/mL。将利福平（Sigma-Aldrich）用作阳性对照，M. abscessus为1000 μg/mL，M. marinum为10 μg/mL。在测量之间，将平板覆盖，放置在铺有湿润纸巾的塑料箱中，在100 rpm的摇床上于37°C（M. abscessus）和28°C（M. marinum）下培养。使用Victor X-3发光计（PerkinElmer）以1秒的积分时间定期测量细菌发光（相对光单位，RLU）。更详细的方案可在protocols.io（6, 7）中找到。参考文献：1. Dalton JP, Uy B, Okuda K, Hall CJ, Denny WA, Crosier PS, Swift S, Wiles S (2017). Screening of anti-mycobacterial compounds in a naturally infected zebrafish embryo model. Journal of Antimicrobial Chemotherapy 72(2):421-427 (doi: 10.1093/jac/dkw421).2. Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible UE, Robertson BD, Wiles S (2010). Optimisation of bioluminescent reporters for use with Mycobacteria. PLOS One. 5(5): e10777 (doi:10.1371/journal.pone.0010777).3. Andreu N, Fletcher T, Krishnan N, Wiles S, Robertson BD (2012). Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence. Journal of Antimicrobial Chemotherapy. 67(2): 404-14 (doi: 10.1093/jac/dkr472). 4. Dalton JP, Uy B, Phummarin N, Copp BR, Denny WA, Crosier PS, Swift S, Wiles S (2016). Effect of common and experimental anti-tuberculosis treatments on Mycobacterium tuberculosis growing as biofilms. PeerJ. 4:e2717 (doi: 10.7717/peerj.2717).5. Andreu N, Zelmer A, Sampson SL, Ikeh M, Bancroft GJ, Schaible UE, Wiles S, Robertson BD (2013). Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase. Journal of Antimicrobial Chemotherapy. 68(9):2118-27 (doi: 10.1093/jac/dkt155).6. Grey A & Wiles S (2021). Bioluminescence-based Minimum Inhibitory Concentration (MIC) testing of pure compounds isolated from fungi against Mycobacterium marinum. Protocols.io. (doi: dx.doi.org/10.17504/protocols.io.3x7gprn). 7. Grey A & Wiles S (2021). Bioluminescence-based Minimum Inhibitory Concentration (MIC) testing of pure compounds isolated from fungi against Mycobacterium abscessus. Protocols.io. (doi: dx.doi.org/10.17504/protocols.io.bumcnu2w).