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  • Discovery and Characterization of the Fully Decorated Nocardiosis-Associated Polyketide Natural Product. Journal of the American Chemical Society Kishore, S., Del Rio Flores, A., Lynch, S. R., Yuet, K. P., Khosla, C. 2024

    Abstract

    The genomes of 40 strains of Nocardia, most of which were associated with life-threatening human infections, encode a highly conserved assembly line polyketide synthase designated as the NOCAP (NOCardiosis-Associated Polyketide) synthase, whose product structure has been previously described. Here we report the structure and inferred biosynthetic pathway of the fully decorated glycolipid natural product. Its structure reveals a fully substituted benzaldehyde headgroup harboring an unusual polyfunctional tail and an O-linked disaccharide comprising a 3-α-epimycarose and 2-O-methyl-α-rhamnose whose installation requires flavin monooxygenase-dependent hydroxylation of the polyketide product. Production of the fully decorated glycolipid was verified in cultures of two patient-derived Nocardia species. In both E. coli and Nocardia spp., the glycolipid was only detected in culture supernatants, consistent with data from genetic knockout experiments implicating roles for two dedicated proteins in installing the second sugar substituent only after the monoglycosyl intermediate is exported across the bacterial cell membrane. With the NOCAP product in hand, the stage is set for investigating the evolutionary benefit of this polyketide biosynthetic pathway for Nocardia strains capable of infecting human hosts.

    View details for DOI 10.1021/jacs.3c13670

    View details for PubMedID 38295028

  • A marine-derived fatty acid targets the cell membrane of Gram-positive bacteria. Journal of bacteriology Upender, I., Yoshida, O., Schrecengost, A., Ranson, H., Wu, Q., Rowley, D. C., Kishore, S., Cywes, C., Miller, E. L., Whalen, K. E. 2023: e0031023

    Abstract

    The rapid evolution of antibiotic resistance is shrinking our stockpile of commercially available antibiotics. Therefore, new antimicrobials with novel mechanisms of action (MoAs) are desperately needed. The free fatty acid, (Z)-13-methyltetra-4-decenoic acid ((Z)-4C-14:1), isolated from a marine sediment bacterium Olleya marilimosa, displays strong inhibition of Gram-positive pathogens with minimal cytotoxic effects to mammalian cells. Here, we applied a combination of experimental approaches to identify the mechanism by which (Z)-4C-14:1 kills the Gram-positive bacterium Bacillus subtilis. Using quick and cost-effective bacterial cytological profiling (BCP), we established the cytological signatures of 17 antibiotics representing 6 general classes of antibiotic MoAs. We used BCP to demonstrate that while (Z)-4C-14:1-treated B. subtilis cells display unique morphological features compared to other antibiotic classes, (Z)-4C-14:1 mode of action shares substantial overlap with the fast-acting antibiotic colistin specifically in Gram-positive cells and daptomycin, both of which target bacterial permeability by destroying the cell membrane and causing extensive cell surface alterations. To further determine if the cell membrane of B. subtilis was the target of (Z)-4C-14:1, we used cell membrane- and peptidoglycan-specific diagnostic stains to investigate bacterial permeability. Our results indicate that (Z)-4C-14:1 destabilizes the cell membrane by pore formation in Gram-positive bacteria and emphasize the importance of mining the marine environment for naturally occurring antibiotics.IMPORTANCEWith the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, new sources of antibiotics that target pathogens of clinical importance are paramount. Here, we use bacterial cytological profiling to identify the mechanism of action of the monounsaturated fatty acid (Z)-13-methyltetra-4-decenoic acid isolated from the marine bacterium Olleya marilimosa with antibacterial effects against Gram-positive bacteria. The fatty acid antibiotic was found to rapidly destabilize the cell membrane by pore formation and membrane aggregation in Bacillus subtilis, suggesting that this fatty acid may be a promising adjuvant used in combination to enhance antibiotic sensitivity.

    View details for DOI 10.1128/jb.00310-23

    View details for PubMedID 37905811

  • Genomic mining and diversity of assembly line polyketide synthases. Open biology Kishore, S., Khosla, C. 2023; 13 (8): 230096

    Abstract

    Assembly line polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for synthesizing many medicinally relevant natural products with remarkable structural variety and biological activity. The decrease in cost of genomic sequencing paired with development of computational tools like antiSMASH presents an opportunity to survey the vast diversity of assembly line PKS. Mining the genomic data in the National Center for Biotechnology Information database, our updated catalogue (https://orphanpkscatalog2022.stanford.edu/catalog) presented in this article revealed 8799 non-redundant assembly line polyketide synthase clusters across 4083 species, representing a threefold increase over the past 4 years. Additionally, 95% of the clusters are 'orphan clusters' for which natural products are neither chemically nor biologically characterized. Our analysis indicates that the diversity of assembly line PKSs remains vastly under-explored and also highlights the promise of a genomics-driven approach to natural product discovery.

    View details for DOI 10.1098/rsob.230096

    View details for PubMedID 37528731

https://orcid.org/0000-0002-6494-4508