Mulberry anthocyanins possess many pharmacological effects including liver protection, anti-inflammation, and anticancer. The aim of this study was to evaluate whether mulberry anthocyanin extract (MAE) exerts beneficial effects against oxidative stress damage in HepG2 cells and Caenorhabditis elegans. In vitro, MAE prevented cytotoxicity, increased glucose consumption and uptake, and eliminated excessive intracellular free radicals in H2O2-induced cells. Moreover, MAE pretreatment maintained Nrf2, HO-1, and p38 MAPK stimulation and abolished upregulation of p-JNK, FOXO1, and PGC-1α that were involved in oxidative stress and insulin signalling modulation. In vivo, extended lifespan was observed in C. elegans damaged by paraquat in the presence of MAE, while these beneficial effects were disappeared in pmk-1 and daf-16 mutants. PMK-1 and SKN-1 were activated after exposure to paraquat and MAE suppressed PMK-1 activation but enhanced SKN-1 stimulation. Our findings suggested that MAE recovered redox status in HepG2 cells and C. elegans that suffered from oxidative stress, which might be by targeting MAPKs and Nrf2.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis. Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane–associated mycolic acid flippase in M. tuberculosis. Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.

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Tuberculosis (TB) remains one of the deadliest infectious diseases, posing a serious threat to global health. Mycobacterium tuberculosis, the causative agent of TB, is an intracellular pathogen that relies on various mechanisms to survive and persist within the host environment. Among the many virulence factors that contribute to pathogenesis, mycobacteria encode Mce systems, putative transporters that are important for lipid uptake. The molecular basis for Mce function(s) is poorly understood. To gain insights into the composition and architecture of mycobacterial Mce systems, we characterized the putative Mce1 complex involved in fatty acid transport. Using affinity purification, we show that the Mce1 system in Mycobacterium smegmatis is an ATP-binding cassette transporter complex comprising YrbE1A/B as heterodimeric transmembrane domains (TMDs) and MceG as nucleotide-binding domains (NBDs), associated with possible heterohexameric assemblies of MCE-domain substrate-binding proteins. We demonstrate that Msmeg_6540 and Mce1A are functionally redundant homologs, likely forming distinct Mce1 complexes that contribute to fatty acid uptake. Finally, we establish that the membrane protein Msmeg_0959, herein renamed to Mce1N, negatively regulates Mce1 function by blocking MceG access to YrbE1B. This regulatory mechanism is conserved in M. tuberculosis Mce1N (Rv0513). Our work offers molecular understanding of Mce complexes, shedding light on lipid metabolism and its regulation in mycobacteria.