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

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

An Engineered Allele of <i>afsQ1</i> Facilitates the Discovery and Investigation of Cryptic Natural Products

New approaches to antimicrobial discovery are needed to address the growing threat of antibiotic resistance. The <i>Streptomyces</i> genus, a proven source of antibiotics, is recognized as having a large reservoir of untapped secondary metabolic genes, many of which are likely to produce uncharacterized compounds. However, most of these compounds are currently inaccessible, as they are not expressed under standard laboratory conditions. Here, we present a novel methodology for activating these “cryptic” metabolites by heterologously expressing a constitutively active pleiotropic regulator. By screening wild <i>Streptomyces</i> isolates, we identified the antibiotic siamycin-I, a lasso peptide that we show is active against multidrug pathogens. We further revealed that siamycin-I interferes with cell wall integrity <i>via</i> lipid II. This new technology has the potential to be broadly applied for use in the discovery of additional “cryptic” metabolites

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