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Identification of Rv3852 as an Agrimophol-Binding Protein in Mycobacterium tuberculosis.

TitleIdentification of Rv3852 as an Agrimophol-Binding Protein in Mycobacterium tuberculosis.
Publication TypeJournal Article
Year of Publication2015
AuthorsZhao N, Sun M, Burns-Huang K, Jiang X, Ling Y, Darby C, Ehrt S, Liu G, Nathan C
JournalPLoS One
Volume10
Issue5
Paginatione0126211
Date Published2015
ISSN1932-6203
KeywordsAnimals, Bacterial Proteins, Carrier Proteins, Humans, Macrophages, Mice, Mycobacterium tuberculosis, Phenols, Structure-Activity Relationship
Abstract

Mycobacterial tuberculosis (Mtb) is able to preserve its intrabacterial pH (pHIB) near neutrality in the acidic phagosomes of immunologically activated macrophages and to cause lethal pathology in immunocompetent mice. In contrast, when its ability to maintain pHIB homeostasis is genetically compromised, Mtb dies in acidic phagosomes and is attenuated in the mouse. Compounds that phenocopy the genetic disruption of Mtb's pHIB homeostasis could serve as starting points for drug development in their own right or through identification of their targets. A previously reported screen of a natural product library identified a phloroglucinol, agrimophol, that lowered Mtb's pHIB and killed Mtb at an acidic extrabacterial pH. Inability to identify agrimophol-resistant mutants of Mtb suggested that the compound may have more than one target. Given that polyphenolic compounds may undergo covalent reactions, we attempted an affinity-based method for target identification. The structure-activity relationship of synthetically tractable polyhydroxy diphenylmethane analogs with equivalent bioactivity informed the design of a bioactive agrimophol alkyne. After click-chemistry reaction with azido-biotin and capture on streptavidin, the biotinylated agrimophol analog pulled down the Mtb protein Rv3852, a predicted membrane protein that binds DNA in vitro. A ligand-protein interaction between agrimophol and recombinant Rv3852 was confirmed by isothermal calorimetry (ITC) and led to disruption of Rv3852's DNA binding function. However, genetic deletion of rv3852 in Mtb did not phenocopy the effect of agrimophol on Mtb, perhaps because of redundancy of its function.

DOI10.1371/journal.pone.0126211
Alternate JournalPLoS One
PubMed ID25978362
PubMed Central IDPMC4433263
Grant ListR01 AI081725 / AI / NIAID NIH HHS / United States

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