title: Profiling Esterases in Mycobacterium tuberculosis Using Far-Red Fluorogenic Substrates.
abstract: Enzyme-activated, fluorogenic probes are powerful tools for studying bacterial pathogens, including Mycobacterium tuberculosis (Mtb). In prior work, we reported two 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO)-derived acetoxymethyl ether probes for esterase and lipase detection. Here, we report four-carbon (C4) and eight-carbon (C8) acyloxymethyl ether derivatives, which are longer-chain fluorogenic substrates. These new probes demonstrate greater stability and lipase reactivity than the two-carbon (C2) acetoxymethyl ether-masked substrates. We used these new C4 and C8 probes to profile esterases and lipases from Mtb. The C8-masked probes revealed a new esterase band in gel-resolved Mtb lysates that was not present in lysates from nonpathogenic M. bovis (bacillus Calmette-Guérin), a close genetic relative. We identified this Mtb-specific enzyme as the secreted esterase Culp1 (Rv1984c). Our C4- and C8-masked probes also produced distinct Mtb banding patterns in lysates from Mtb-infected macrophages, demonstrating the potential of these probes for detecting Mtb esterases that are active during infections.
authors.affiliation: Program in Chemical Biology and the Department of Biomedical Engineering, Oregon Health & Science University , Portland, Oregon 97201, United States.
authors.forename: Katie R
authors.initials: KR
authors.lastname: Tallman
authors.affiliation: Program in Chemical Biology and the Department of Biomedical Engineering, Oregon Health & Science University , Portland, Oregon 97201, United States.
authors.forename: Samantha R
authors.initials: SR
authors.lastname: Levine
authors.affiliation: Program in Chemical Biology and the Department of Biomedical Engineering, Oregon Health & Science University , Portland, Oregon 97201, United States.
authors.forename: Kimberly E
authors.initials: KE
authors.lastname: Beatty
mesh_terms: D004950:Esterases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D004950:Esterases