Isolation And Mechanistic Study Of The Antifungal Polycyclic Tetramate Macrolactams From Lysobacter Enzymogenes C3

The incidence of fungal infections, especially the clinical Candida albicans infection, has increased due to the misuse of antibiotics, radiation, immunosuppressant treatment and the use of various built-in medical apparatus and instruments. In addition, the overreliance on a few traditional drugs for the treatment of C. albicans infection has led to the emergence of persistence, which makes the therapy of clinical C. albicans much more difficult. It is imperative to identify new anti-Candida drugs with distinct chemical structures and novel mode of actions.Microorganisms are one of the most important sources of bioactive compounds. Traditionally, Gram-positive bacteria and fungi are the focus of bioactive natural product isolation, and the Gram-negative bacteria are largely ignored although they are also rich in natural products. In recent years, a series of active compounds with novel structural features have been obtained, which opens a new resource for active natural product discovery. In this study, we investigated the secondary metabolites from Lysobacter enzymogenes C3 and obtained a series of antifungal compounds, which belong to PTMs. Furthermore, we studied these PTMs' antifungal mechanism.In the first chapter, we reviewed the current status of fungal infections and the current antifungal drugs in clininal use. These drugs are limited in terms of chemistry and targets, which mainly focus on cell walls and cell membranes. Lysobacters species represent a new and rich source for discovering new antifungal compounds. We also review the development of the Lysobacters.In the second chapter, we described the screening of the culture media for Lysobacter enzymogens C3. We found that TSB medium produced the highest output of natural products. The results showed that 1/10 TSB consistently gave a high yield of the antifungal compounds. Using this medium, we were able to isolate four compoundsfrom L. enzymogenes C3. Structural determination using MS and NMR revealed that these four compounds belong to PTMs, including one new PTM (alteramide B) and three known PTMs (HSAF,3-deOH-HSAF and 3-deOH-alteramide B).In the third chapter, we described the investigation of the antifungal mechanism of HSAF. We first used Magnaporthe grisea as the model fungus and found that HSAF treatment could cause to apoptosis through the analysis of differential transcription. In the following studies, we used C. albicans as the test fungus for the mechanism of HSAF. The cell wall has a relative complex outer layer and a dense inner structure and affects the small material transporting into the cell. To elucidate the underlying mechanism of HSAF against fungi, the C. albicans protoplast was prepared. We applied the DCFH-DA assay to measure the levels of ROS in C. albicans upon HSAF treatment. The activity of HSAF was decreased after adding reductive agents such as ascorbic acid, thiourea, N-acetyl-L-cysteine and glutathione. The results indicate that HSAF inhibits fungal growth through the ROS accumulation. Using the analysis of biological means, we found HSAF could induce the ROS accumulation, trigger collapse of MMP, arrest the cell cycle at the G2/M phase and trigger apoptosis.In the fourth chapter, the mechanism of alteramide B (ATB), a new PTM compound, was studied. ATB displayed evident inhibitory effect on the yeast and germ tube formation of C. albicans. ATB induced the ROS accumulation and triggered collapse of mitochondrial membrane potential. ATB arrested the cell cycle at the G2/M phase and led to apoptosis. The anti-Candida activity of ATB including the growth inhibition of budding yeast and germ tube was strongly reduced by the addition of AA. These results indicated that the anti-Candida activity of ATB was mediated by ROS production. We further searched for the potential target of ATB in C. albicans. Since ATB could cause changes in the inner mitochondrial membrane potential and dynamic mitochondrial disorders are associated with tubulins, we investigated the potential interaction between ATB and tubulins. ATB bound to tubulins as detected by immunofluorescence and CETSA in HeLa cells; we extracted the tubulins from porcine and studied the interaction of ATB with tubulins in vitro. To gain further insight into the binding interaction of ATB with tubulins, the molecular dynamics studies of ATB binding to tubulins were performed. We found that ATB could physically interact with ?-tubulin. Molecular dynamics studies demonstrated that ATB selectively occupies the twelve key residues binding site of ?-tubulin. The ?-tubulin was probably the target of ATB in C. albicans cells, and L215, L217, L273, L274 and R282 may be the binding site for ATB, which was consistent with the molecular modeling results. Encouraged by the potent in vitro anti-Candida activity of ATB, we went on to evaluate in vivo anti-Candida activity of ATB in a BALB/c mice model. ATB exhibited a similar efficacy as amphotericin B against C. albicans in vivo.In summary, we identified a new antifungal compound ATB from L. enzymogenes C3 and studied the mechanism of HSAF and ATB. The results set up the foundation for the further study of the antifungal mechanism of other compounds in the PTM family. ATB may represent a promising lead compound for the development of new antifungal drugs to treat invasive infections caused by C. albicans.