Marine Streptomyces-derived 2,4-di-tert-butylphenol enhances autophagy to eliminate mycobacteria in human macrophages

Abstract

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), remains a major global health threat, highlighting the need for novel therapeutic strategies. Host-directed therapies (HDTs), particularly autophagy induction via natural or pharmacological agents, offer promising strategies for combating mycobacteria. This study assessed the anti-mycobacterial potential of marine-derived Streptomyces spp. Briefly, the isolated marine Streptomyces spp. underwent primary and secondary screenings to assess their inhibitory activity against M. smegmatis. Notably, the cell-free extract of Streptomyces fradiae DNS4 (S. fradiae DNS4) demonstrated the most vigorous anti-mycobacterial activity. Optimization of culture conditions revealed maximal secondary metabolite production in ISP-7 medium at 28°C and pH 7 by S. fradiae DNS4. Furthermore, extraction using ethyl acetate, followed by GC-MS analysis, identified seventeen compounds, including the potent anti-mycobacterial secondary metabolite 2,4-di-tert-butylphenol [2,4-(DTBP)]. Subsequent experiments evaluated the host-directed anti-mycobacterial mechanism of action for 2,4-(DTBP) using dTHP-1 cells. At a non-cytotoxic concentration (10 μM), 2,4-(DTBP) treatment markedly reduced intracellular mycobacterial survival compared to untreated controls. Multiparametric investigations into autophagy induction, including LC3-I to LC3-II conversion, protein expression profiling of key autophagy markers, and MDC staining, demonstrated enhanced autophagic responses in 2,4-(DTBP)-treated macrophages. Upon 2,4-(DTBP) treatment, enhanced autophagic flux was further confirmed by increased LC3-II accumulation under Baf-A1 pre-treatment conditions. Importantly, autophagy inhibition by pre-treatment with 3-MA abolished the observed anti-mycobacterial effects, confirming the specific autophagy-dependent mechanism of 2,4-(DTBP). Collectively, these findings highlight the potential of marine-derived 2,4-(DTBP) from S. fradiae DNS4 as a novel host-directed anti-mycobacterial agent, which is mediated primarily through the targeted augmentation of autophagy in human macrophages.