Browsing by Author "Rubin, Eric J"

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    Open Access
    High-throughput sequencing enhanced phage display identifies peptides that bind mycobacteria
    (Public Library of Science, 2013) Ngubane, Nqobile A C; Gresh, Lionel; Ioerger, Thomas R; Sacchettini, James C; Zhang, Yanjia J; Rubin, Eric J; Pym, Alexander; Khati, Makobetsa
    Bacterial cell wall components have been previously used as infection biomarkers detectable by antibodies. However, it is possible that the surface of the Mycobacterium tuberculosis ( M. tb ), the causative agent of tuberculosis (TB), also possesses molecules which might be non-antigenic. This makes the probing of biomarkers on the surface of M. tb cell wall difficult using antibodies. Here we demonstrate the use of phage display technology to identify peptides that bind to mycobacteria. We identified these clones using both random clone picking and high throughput sequencing. We demonstrate that random clone picking does not necessarily identify highly enriched clones. We further showed that the clone displaying the CPLHARLPC peptide which was identified by Illumina sequencing as the most enriched, binds better to mycobacteria than three clones selected by random picking. Using surface plasmon resonance, we showed that chemically synthesised CPLHARLPC peptide binds to a 15 KDa peptide from M.tb H37Rv whole cell lysates. These observations demonstrate that phage display technology combined with high-throughput sequencing is a powerful tool to identify peptides that can be used for investigating potential non-antigenic biomarkers for TB and other bacterial infections.
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    Open Access
    Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis
    (Public Library of Science, 2013) Ioerger, Thomas R; O'Malley, Theresa; Liao, Reiling; Guinn, Kristine M; Hickey, Mark J; Mohaideen, Nilofar; Murphy, Kenan C; Boshoff, Helena I M; Mizrahi, Valerie; Rubin, Eric J
    Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis , especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery.