title: Identification of Rv3852 as an Agrimophol-Binding Protein in Mycobacterium tuberculosis.
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.
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhao
authors.affiliation: Tsinghua-Peking Center for Life Sciences and Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, P. R. China.
authors.forename: Mingna
authors.initials: M
authors.lastname: Sun
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Kristin
authors.initials: K
authors.lastname: Burns-Huang
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Xiuju
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ling
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Crystal
authors.initials: C
authors.lastname: Darby
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Sabine
authors.initials: S
authors.lastname: Ehrt
authors.affiliation: Tsinghua-Peking Center for Life Sciences and Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, P. R. China.
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Carl
authors.initials: C
authors.lastname: Nathan
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000378:metabolism* / Q000502:physiology; D010636:Phenols / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D010636:Phenols; C022779:agrimophol