New liposidomycin congeners produced by *Streptomyces* sp. TMPU-20A065, anti-*Mycobacterium avium* complex agents with therapeutic efficacy in a silkworm infection model

Mycobacterium avium complex (MAC) infections, mainly due to M. avium and M. intracellulare, are increasing in developed countries and have symptoms similar to tuberculosis but with slower progression and poorer prognosis. Current first-line therapy includes clarithromycin combined with rifampicin and ethambutol, but efficacy is often insufficient, requires long-term administration (>1 year), and can lead to drug resistance. Although amikacin liposome inhalation suspension was approved in 2018, its use is limited to cases refractory to conventional therapy. Therefore, there is an urgent need for new anti-MAC agents with novel structures and mechanisms. The authors developed an in vivo-mimic silkworm infection model for primary screening of anti-MAC agents and applied it to microbial resources, leading to the identification of three new liposidomycin congeners (1, 2, and 4) and 14 known liposidomycins (3, 5–17) from Streptomyces sp. TMPU-20A065. The study reports the fermentation, isolation, structural elucidation, and in vitro and in vivo antimycobacterial activities of these liposidomycins.

General analytical methods: UV spectra (Hitachi U-3310), IR spectra (JASCO FT/IR-4100), optical rotations (JASCO P-2300), HRFAB-MS (JEOL JMS-MS 700), and NMR (JEOL JNM-ECZ600R/S1) were used for structural elucidation. Microorganisms: For antimycobacterial assays: Mycobacterium avium JCM 15430, M. intracellulare JCM 6384, M. bovis BCG Pasteur, and M. smegmatis NBRC 3207. For antibacterial assays: Bacillus subtilis NBRC 3134, Staphylococcus aureus NBRC 13276, Escherichia coli NBRC 3972, and Pseudomonas aeruginosa NBRC 13275. Producing strain and identification: Liposidomycin-producing actinomycete TMPU-20A065 was isolated from soil collected in Yamaguchi city, Yamaguchi, Japan, and identified as Streptomyces sp. by 16S rDNA sequence BLAST. Fermentation: Agar plate culture on ISP medium No. 2 was used to inoculate seed medium (potato starch 2.4%, yeast extract 0.5%, glucose 0.1%, peptone 0.3%, Ehrlich meat extract 0.3%, CaCO3 0.4%, pH 7.0) in 500-ml flasks (100 ml), shaken at 180 rpm, 27 °C for 3 days. Seed culture (1.0 ml) was transferred into 180 flasks (500-ml) each with 200 ml production medium (glucose 0.5%, Solulys 0.5%, KH2PO4 0.5%, MgSO4·7H2O 0.5%, oatmeal 0.5%, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, CoCl2·6H2O each 0.001%; pH 7.0). Fermentation at 180 rpm, 27 °C for 7 days. Isolation and purification: The 7-day culture broth (36 L) was centrifuged; supernatant (14 L × 2) was adsorbed on Diaion HP-20 and eluted stepwise with 0%, 50%, 100% MeOH and 50% acetone. The 50% acetone fraction was concentrated, lyophilized (2.44 g), then fractionated by MPLC on ODS with a CH3CN-0.05% TFA gradient. Active fractions were purified by preparative HPLC on ODS and specialty columns (PEGASIL ODS SP100, Discovery HS F5, COSMOSIL nap) using isocratic and gradient CH3CN-0.05% TFA mobile phases with UV detection at 210 nm to yield compounds 1–17. Yields included: 1 (1.12 mg), 2 (7.65 mg), 3 (0.20 mg), 4 (1.21 mg), 5 (3.65 mg), 6 (3.35 mg), 7 (0.33 mg), 8 (1.49 mg), 9 (3.17 mg), 10 (0.97 mg), 11 (0.75 mg), 12 (0.60 mg), 13 (1.11 mg), 14 (0.39 mg), 15 (0.22 mg), 16 (0.28 mg), 17 (0.51 mg). Structural elucidation: Known compounds 3 and 5–17 were assigned by comparison of NMR and MS data with literature (liposidomycins Y-III, Z-I, A-I, Z-III, B-I, C-I, A-III, B-III, C-III, isoH-I, G-I, K-III, M-I, M-III). New compounds 1, 2, and 4 exhibited UV maxima at ~202–203 and 263–265 nm; IR bands consistent with alcohol, alkyl, and carbonyl groups; and HR-FAB-MS supporting formulas C40H63N5O21S (1), C42H63N5O21S (2), C42H65N5O21S (4). 1H/13C NMR and 2D NMR (COSY, etc.) in CD3OD established a type-I liposidomycin core containing 5′-substituted uridine, 5-amino-5-deoxyribose-2-sulfate, perhydro-1,4-diazepine, and 3-methylglutaric acid, with differing acyl side chains: 1 bears a decanoic acid moiety; 2 bears tetradec-5,8-dienoic acid with both double bonds in cis configuration (J5a-6a ≈ 11.0 Hz by decoupling); 4 bears 7-tetradecenoic acid. In vitro antimycobacterial assay (broth microdilution): Mycobacteria grown in Middlebrook 7H9 broth (with Tween 80, BSA, glucose, NaCl) at 37 °C for 2–7 days to ~1.0×10^9 CFU ml⁻¹; diluted 1:500; 95 µl inoculum plus 5 µl sample per well in 96-well plates; incubated 37 °C for 2–7 days; turbidity at 550 nm; MIC defined as lowest concentration inhibiting 90% of control growth. In vivo-mimic silkworm infection model: Fifth-instar Bombyx mori larvae (~2 g) infected by hemolymph injection of M. avium or M. intracellulare (2.5×10^7 CFU larva⁻¹ g⁻¹ in 50 µl 7H9); within 30 min, test samples (50 µl in saline or 10% DMSO) injected; larvae maintained at 37 °C without feed; survival monitored for 96 h. ED50 defined as dose achieving 50% survival normalized per 1 g larva. Antibacterial assay (non-mycobacterial): CLSI M07-A09-compliant broth microdilution in Mueller-Hinton broth at 37 °C for 24 h; inoculum prepared to OD550 0.0548 (~10^8 CFU ml⁻¹), diluted 1:3000; MIC determined at 550 nm as 90% inhibition threshold.

- Three new liposidomycin congeners (1, 2, 4) and 14 known liposidomycins (3, 5–17) were isolated from Streptomyces sp. TMPU-20A065. - Structural elucidation (NMR, HR-FAB-MS) showed all three new compounds are type-I liposidomycins featuring sulfate groups and methylglutaric acid, differing in acyl side chains: 1 contains a decanoic acid moiety; 2 contains tetradec-5,8-dienoic acid with cis double bonds; 4 contains 7-tetradecenoic acid. - In vitro activity: Compounds 1–17 exhibited antimycobacterial activity against M. avium and M. intracellulare with MICs ranging from 2.0 to 64 µg ml⁻¹. - In vivo-mimic efficacy: Infected silkworm model ED₅₀ values ranged from 0.12 to 3.7 µg larva⁻¹ g⁻¹, indicating potent therapeutic effects across the series. - Known congeners were assigned as liposidomycins Y-III (3), Z-I (5), A-I (6), Z-III (7), B-I (8), C-I (9), A-III (10), B-III (11), C-III (12), isoH-I (13), G-I (14), K-III (15), M-I (16), and M-III (17) by literature comparison.

The study addresses the need for novel anti-MAC agents by discovering and characterizing new liposidomycin congeners with demonstrated activity in both in vitro and in vivo-mimic models. The integration of a silkworm infection model into primary screening enabled assessment of therapeutic efficacy beyond growth inhibition, yielding compounds with ED50 values indicative of in vivo potential. Structural diversity in the acyl side chains among the type-I liposidomycins was defined, and all isolated compounds showed activity against key MAC pathogens (M. avium, M. intracellulare), supporting liposidomycins as promising leads for MAC treatment.

This work identifies three new type-I liposidomycin congeners (1, 2, 4) and 14 known congeners from Streptomyces sp. TMPU-20A065, elucidates their structures, and demonstrates their anti-MAC activities in vitro (MIC 2.0–64 µg ml⁻¹) and therapeutic efficacy in a silkworm infection model (ED₅₀ 0.12–3.7 µg larva⁻¹ g⁻¹). The findings highlight liposidomycins as potential candidates for further development against MAC infections and showcase the utility of an in vivo-mimic silkworm model for early-stage anti-mycobacterial screening.