Regulation of specialised metabolites in Actinobacteria – Expanding the paradigms

The increase in availability of actinobacterial whole genome sequences has revealed huge numbers of specialised metabolite biosynthetic gene clusters, encoding a range of bioactive molecules such as antibiotics, antifungals, immunosuppressives and anticancer agents. Yet the majority of these clusters are not expressed under standard laboratory conditions in rich media conditions. Emerging data from studies of specialised metabolite biosynthesis suggest that the diversity of regulatory mechanisms is greater than previously thought and these act at multiple levels, through a range of signals such as nutrient limitation, intercellular signalling and competition with other organisms. Understanding the regulation and environmental cues that lead to the production of these compounds allows us to identify the role which these compounds play in their natural habitat as well as providing tools to exploit this untapped source of specialised metabolites for therapeutic uses. Here we provide an overview of novel regulatory mechanisms that act in physiological, global, and cluster specific regulatory manners on biosynthetic pathways in Actinobacteria and consider these alongside their ecological and evolutionary implications

An oxindole efflux inhibitor potentiates azoles and impairs virulence in the fungal pathogen Candida auris

Candida auris is an emerging fungal pathogen that exhibits resistance to multiple drugs, including the most commonly prescribed antifungal, fluconazole. Here, we use a combinatorial screening approach to identify a bis-benzodioxolylindolinone (azoffluxin) that synergizes with fluconazole against C. auris. Azoffluxin enhances fluconazole activity through the inhibition of efflux pump Cdr1, thus increasing intracellular fluconazole levels. This activity is conserved across most C. auris clades, with the exception of clade III. Azoffluxin also inhibits efflux in highly azole-resistant strains of Candida albicans, another human fungal pathogen, increasing their susceptibility to fluconazole. Furthermore, azoffluxin enhances fluconazole activity in mice infected with C. auris, reducing fungal burden. Our findings suggest that pharmacologically targeting Cdr1 in combination with azoles may be an effective strategy to control infection caused by azole-resistant isolates of C. auris.U01 TR002625 - NCATS NIH HHS; MOP-133636 - CIHR; U19 AI110818 - NIAID NIH HHS; R35 GM118173 - NIGMS NIH HHS; FDN-154288 - CIHR; R01 AI141202 - NIAID NIH HHS; R01 AI073289 - NIAID NIH HHSPublished versio

Exploiting diverse chemical collections to uncover novel antifungals

The rise in drug resistance amongst pathogenic fungi, paired with the limited arsenal of antifungals available is an imminent threat to our medical system. To address this, we screened two distinct compound libraries to identify novel strategies to expand the antifungal armamentarium. The first collection wasthe RIKEN Natural Product Depository (NPDepo), which was screened for antifungal activity against four major human fungal pathogens: Candida albicans, Candida glabrata, Candida auris, and Cryptococcus neoformans. Through a prioritization pipeline, one compound, NPD6433, emerged as having broad-spectrum antifungal activity and minimal mammalian cytotoxicity. Chemical-genetic and biochemical assays demonstrated that NPD6433 inhibits the essential fungal enzyme fatty acid synthase 1 (Fas1). Treatment with NPD6433 inhibited various virulence traits in C. neoformans and C. auris, and rescued mammalian cell growth in a co-culture model with C. auris. The second compound library screened was adiversity-oriented collectionfrom Boston University. This chemical screen was focused on identifying novel molecules that enhance the activity of the widely deployed antifungal, fluconazole, against C. auris. Through this endeavour, we discovered a potent compound that enhanced fluconazole efficacy against C. auris through increasing azole intracellular accumulation. This activity was dependent on expression of the multidrug transporter geneCDR1, suggesting that this compound targets efflux mechanisms. Furthermore, this molecule significantly reduced fungal burden alone and in combination with fluconazole in a murine model of C. auris disseminated infection. Overall, this work identifies novel compounds with bioactivity against fungal pathogens, revealing important biology, and paving the way for the critical development of therapeutic strategies.Published versio

Crystallographic and biochemical characterization of key steps in reductasporine and capuramycin biosynthesis

Studying individual biosynthetic transformations in the creation of natural products often reveals surprising and powerful chemical reactivities and molecular handling strategies. Indolocarbazole bisindoles have been widely tested in clinical studies and arise from oxidative dimerization of L-tryptophan. Among bisindoles, reductasporine bears an unusual dimethylpyrrolinium structure. Its biosynthesis differs from other indolocarbazole pathways by two tailoring enzymes: the imine reductase RedE and N,N-dimethyltransferase RedM. Here I reconstitute this pathway in vitro and show that RedE prevents unstable indolocarbazole intermediates from becoming oxidized and provides reduced didemethylreductasporine substrate to RedM. Employing X-ray crystallography, I solved two ternary complexes of RedE co-crystallized with the substrate-mimic arcyriaflavin A, revealing an extended active site cleft with distinct secondary indolocarbazole binding site. Site-directed mutagenesis confirms the conserved active site aspartate (D168) is essential for activity and anchors the substrate via hydrogen-bonding. Variants targeting the secondary binding site reduce catalytic efficiency, suggesting this site protects the substrate from autooxidation. I solved the 1.7 Å structure of RedM demonstrating it adopts distinct open and closed conformations with either SAH or SAM cofactor, respectively. Site-directed mutagenesis, docking and sequence bioinformatics identify conserved substrate-recognizing residues and suggest dimethyltransferase catalytic activity likely arises from precise orientation and desolvation of the substrate. Recently, Cap15 has been shown to be an oxygen- and pyridoxal phosphate (PLP)-dependent enzyme and is the first example of this activity in the L-seryl-tRNA(Sec) selenium transferase enzyme family. It catalyzes oxidative-decarboxylation of 5ʹ-glycyl uridine to the corresponding 5ʹ-carboxamide uridine. Solving for the 2.40 Å resolution crystal structure of Cap15 shows PLP bonds to K230 and a phosphate anion in the active site bridges N- and C-terminal domains. Sequence analysis reveals the loop proximal to the internal aldimine and hydrogen bonding to the active site phosphate is strictly conserved among Cap15 homologues present in capuramycin-type gene clusters. The crystal structures provide a basis for further investigations into the sequence-determinants of secondary binding site formation in RedE, dimethylation activity in RedM and oxygen-consuming activity in Cap15. RedE in particular can serve as a starting-point in the engineering of imine reductases to accommodate large substrates in the production of industry-relevant chiral amines.Science, Faculty ofChemistry, Department ofGraduat

Uncovering ‘Cryptic’ Natural Products from Streptomyces Bacteria

The Streptomyces bacteria produce natural products that have potent biological activity against other organisms: 60% of our antibiotics are derived from this source. Genome sequencing reveals genes for many more. One view in the field is that these “cryptic” metabolites could serve as badly needed antibiotics for antibiotic resistant infections. However, many of them are produced at too low yields for structural and mechanistic characterization. The top priority is finding broadly applicable approaches to enhancing the yields of these molecules. To address this, I took advantage of a conserved regulator of specialized metabolism to develop a generally applicable tool, called AfsQ1*, that heterologously induces many specialized metabolic genes. Using this technology, I developed two antibiotic discovery screens. First, I identified afsQ1*-induced antibacterial activities and purified the active agents. This led to the discovery of the antibiotic siamycin-I, a potent inhibitor of antibiotic resistant Gram-Positive bacteria. I demonstrated that this compound targets the lipid-II component of cell wall biogenesis, the first of this class of molecules to do so. The second approach used comparative metabolomics to identify afsQ1*-induced novel masses. By NMR I identified a new pepticinnamin analogue, whose family are inhibitors of an eukaryotic post-translational modification called farnesylation. Farnesyl transferase inhibitors were previously investigated (unsuccessfully) as anticancer medicines. I demonstrate, however, that they are candidates for antifungal therapy, because they block morphological switching- a key virulence trait in lower fungi. This work suggests a new paradigm in antifungal therapy. Finally, I dissect an interaction between Lactobacillus reuteri and C. albicans. When co-cultured together C. albicans cannot undergo filamentous growth and I found that this inhibition is molecule-mediated. 1-acetyl β-carboline is the active metabolite isolated from L. reuteri and I found that its production is prevalent throughout the Lactobacillus genera. I also synthesized a new β-carboline analogue for future clinical trials.Ph.D.2021-06-22 00:00:0