Sterling Sandler
New York University
Disruption of Mycobacterial DnaK Synergizes with Miscoding Agents
Molecular chaperone systems play crucial roles in maintaining proteostasis through protein folding, disaggregation, and refolding. In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, the molecular chaperone DnaK is hypothesized to be essential for cell growth likely due to its role in nascent protein folding. Previous work has shown that telaprevir (TP), an FDA-approved viral peptidomimetic protease inhibitor is an allosteric inhibitor of Mtb DnaK ATPase. In addition to inhibiting DnaK in vitro, TP was shown to disrupt proteostasis in cells by enhancing cellular sensitivity to heat, select aminoglycoside antibiotics and by decreasing frequency of resistance to rifampin in mycobacteria. We aimed to use TP to probe to consequences of chaperone inhibition and other stressors in pathogenic bacteria. We hypothesized that chaperone inhibition would synergize with specific classes of antibiotic that disrupt proteostasis. We utilized cell-based assays with 15 antibiotics and found telaprevir potentiates protein synthesis inhibitors Mycolicibacterium smegmatis. Checkerboard assays in Mtb illustrated that telaprevir is synergistic with miscoding agents. The effect of the aminoglycoside and chaperone inhibition was further probed with proteomics to identify the cellular response to dual inhibition in addition to an analysis of the translation error landscape. We then utilized essential chaperonin GroEL2 as a reporter for missense mutation solubility and DnaK association. Taken together, our work confirms the role of DnaK in handling mistranslated proteins and supports the rationale for studying molecular chaperones for adjuvant therapy.
Molecular chaperone systems play crucial roles in maintaining proteostasis through protein folding, disaggregation, and refolding. In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, the molecular chaperone DnaK is hypothesized to be essential for cell growth likely due to its role in nascent protein folding. Previous work has shown that telaprevir (TP), an FDA-approved viral peptidomimetic protease inhibitor is an allosteric inhibitor of Mtb DnaK ATPase. In addition to inhibiting DnaK in vitro, TP was shown to disrupt proteostasis in cells by enhancing cellular sensitivity to heat, select aminoglycoside antibiotics and by decreasing frequency of resistance to rifampin in mycobacteria. We aimed to use TP to probe to consequences of chaperone inhibition and other stressors in pathogenic bacteria. We hypothesized that chaperone inhibition would synergize with specific classes of antibiotic that disrupt proteostasis. We utilized cell-based assays with 15 antibiotics and found telaprevir potentiates protein synthesis inhibitors Mycolicibacterium smegmatis. Checkerboard assays in Mtb illustrated that telaprevir is synergistic with miscoding agents. The effect of the aminoglycoside and chaperone inhibition was further probed with proteomics to identify the cellular response to dual inhibition in addition to an analysis of the translation error landscape. We then utilized essential chaperonin GroEL2 as a reporter for missense mutation solubility and DnaK association. Taken together, our work confirms the role of DnaK in handling mistranslated proteins and supports the rationale for studying molecular chaperones for adjuvant therapy.