Antibiotic resistance has been and remains a major problem in our society. The main solution to this problem is to search and study the mechanisms of antibiotic action. Many groups of secondary metabolites, including antimicrobial ones, are produced by the Actinomycetota phylum. The actinobacterial strains isolated from habitats that have not been well studied are of great interest. Due to high resource competition, antibiotics are now considered a ‘trump card in the game of life’ due to their presence in natural substrates with limited nutrients. Potentially, strains isolated from such habitats can be producers of novel or poorly studied antibiotics. In the current research, we identified the strain Streptomyces sp. AP22 from the soils of the Akhshatyrsky Gorge, which is capable of producing pentalenolactone. This study describes the phenotypic and morphological characteristics of Streptomyces sp. AP22 and its biological activity. Pentalenolactone is a known inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important enzyme involved in glycolysis. We identified a previously unknown mutation in the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase that confers resistance to this antibiotic compound. This antibiotic is not used in clinical practice, so its application as a selectable marker will not lead to the creation of pathogens resistant to clinically relevant antibiotics. In this case, the selectable marker is based on a genetic construct containing the glyceraldehyde-3-phosphate dehydrogenase gene with a resistance mutation. The use of this selectable marker can be applied to various genetic and molecular techniques, such as cloning and transformation. This can help to facilitate genetic and molecular biology studies of strains resistant to standard selectable markers such as kanamycin or ampicillin.

Antimicrobial resistance is a major threat to modern society and healthcare, as it severely compromises the efficacy of standard antibiotic treatments. To meet the ever-increasing demand for novel antimicrobial drugs, it is crucial to develop new strategies for screening antimicrobial compounds and improve existing high-throughput techniques. Reporter systems that employ specific genetic markers are powerful tools not only for detecting antimicrobial activity of the substance being studied, but also for identifying the potential mechanism of its action. Among other metabolic pathways, RNA biosynthesis machinery is considered a promising molecular target as it remains underutilized in current antimicrobial therapy and therefore is rarely exposed to drug pressure. However, there is no suitable biomarker for identifying compounds that inhibit the transcription in Gram-negative bacteria. Combining bioinformatic search and RT-qPCR experimental validation, we have established the overexpression of the spermidine synthase gene (speE) as a biomarker associated with impaired transcription in Escherichia coli. Monitoring the expression level of speE in antibiotic-treated cells enables reliable detection of compounds that inhibit bacterial RNA-polymerase, such as rifampicin and fidaxomicin. Moreover, our screening system was successfully applied in practice to analyze chromatography fractions from fermentation broth of antibiotic producers, with compounds of the rifamycin family being identified as hits and isolated. The proposed method has the potential to be used in sequential screening procedures to reveal active antimicrobial compounds that inhibit bacterial transcription process, giving the world novel antimicrobials with minimal risk of resistance development.

Background/Objectives: Autophagy is an important cellular self-cleansing process whose normal functioning is essential for preventing many age-related diseases. The search for and study of new autophagy activators allows the proposal of novel therapeutic approaches for the treatment of age-related diseases. Medical plants are a rich source of bioactive compounds with variable functions. In this study, we propose an HR-LCMS/MS-based technique for identifying the principal autophagy activators in plant extracts. Methods: We performed a Western blot analysis of the autophagy-inducing activity of plant extract HPLC fractions on a model of SH-SY5Y cells. The composition of the fractions showing autophagy-activating potential was determined via HR-LCMS/MS. Results: We analyzed five plants known to produce autophagy activators and proved the ability of the method to detect the main bioactive compounds. Additional screening demonstrated for the first time that Astragalus dasyanthus is a producer of the autophagy-inducer glabrol. Conclusions: The described HR-LCMS/MS-based method for identifying autophagy activators in multicomponent plant extracts is effective and could be used for further high-throughput screening.

Autophagy is a conserved lysosome-dependent process essential for cellular homeostasis, and pharmacological activation of autophagy has emerged as a promising therapeutic strategy for age-related, neurodegenerative, metabolic, and malignant diseases. Despite the growing interest in natural autophagy modulators, lichens remain largely unexplored as a source of autophagy-inducing compounds. Here, we tested ethanolic extracts from four commercially available lichens using LC3-II accumulation as a primary readout. Usnea sp. was identified as the most active species. Activity-guided fractionation followed by HRMS and NMR analyses identified salazinic acid as the active metabolite. Salazinic acid increased LC3-II accumulation, enhanced autophagic flux in the presence of bafilomycin A1, and promoted autophagosome formation as confirmed by transmission electron microscopy. The compound exhibited low cytotoxicity in human cell lines at concentrations exceeding those required for autophagy induction. Mechanistically, salazinic acid activated the phosphorylation of AMPK, ULK1, and AKT; however, it retained its ability to stimulate autophagic flux in AMPK- and ULK1-deficient cells, indicating that its activity is not strictly dependent on the canonical AMPK–ULK1 signaling pathway. Collectively, our findings identify salazinic acid as a previously unrecognized inducer of productive autophagic flux and demonstrate that lichen secondary metabolites represent a promising and largely untapped source of novel autophagy-modulating compounds.