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An Allosteric Inhibitor of Bacterial Hsp70 Chaperone Potentiates Antibiotics and Combats Resistance

ABSTRACT: In the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), DnaK and its cofactors are proposed antimycobacterial targets. Here, we discover that a repurposed drug called telaprevir is able to allosterically inhibit the ATPase activity of DnaK from several organisms, and prevents chaperone function by mimicking peptide substrates. We designed a photoreactive probe that binds to the same region of DnaK as telaprevir. After crosslinking the probe, mass spectrometry was used to identify the site of interaction. Based on crosslink spectral matches (CSMs), eighty percent of the modified peptides were located in the beta-sandwich domain spanning from Glu398 to Ser409. The most highly represented modified residue was Asn406, followed by Glu398 and Asp405, all of which are present in the peptide binding cleft.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mycobacterium Tuberculosis (ncbitaxon:1773)

SUBMITTER: Beatrix Ueberheide

PROVIDER: MSV000088127 | MassIVE |

REPOSITORIES: MassIVE

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Project description:Human infection with Mycobacterium tuberculosis results in a continuum of ill-defined, clinical manifestations with stable latent M. tuberculosis infection (LTBI) and severe active disease at the ends. Identifying different states of infection is of importance to tuberculosis (TB) control since risk of developing active disease varies among different asymptomatic states while infectiousness varies among patients with different bacterial burden. We investigated changes in proteome-scale antibody responses during disease progression in a non-human primate model of tuberculosis. We probed M. tuberculosis proteome microarrays with serial sera collected from three infection-outcome groups (active, reactivation, and latent). We found that each infection outcome is associated with characteristic changes in the antibody levels and number of antigenic targets, which suggested an association between antibody responses and bacillary burden. Additional proteome-scale serological profiling of > 400 human TB suspects established that antibody responses are positively associated with bacterial load. Thus tuberculosis-specific antibody levels and number of antigenic targets increases with disease progression. Serum samples collected from adult patients with suspected tuberculosis during a multi-site study was used to probe whole proteome microarrays. Subject recruitment was conducted under uniform protocols approved by the institutional ethics committee at each site. Final diagnosis of active TB was based on positive M. tuberculosis culture results. The active TB patients were further subdivided into smear-positive and negative disease based on results of Ziehl-Neelsen staining of sputum smears for acid fast bacilli. Active TB was excluded as a diagnosis (Non-TB Disease [NTBD] patients) based on having negative M. tuberculosis culture and smear results and on having an alternate diagnosis. All subjects were presumably negative for HIV infection given the very low incidence of HIV infection in the study sites. Sera from 169 TB and 242 NTBD patients were selected for microarray probing. The control sera (n = 14) which was used to generate negative control distribution for each protein were negative to latent M. tuberculosis infection, as indicated by negative results to tuberculin skin test.

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An Allosteric Inhibitor of Bacterial Hsp70 Chaperone Potentiates Antibiotics and Combats Resistance

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