RESUMEN
Mycobacterium tuberculosis (Mtb) is the deadliest bacterial pathogen in the world. An estimated one-third of humans harbor Mtb in a dormant state. These asymptomatic, latent infections impede tuberculosis eradication due to the long-term potential for reactivation. Dormant Mtb has reduced enzymatic activity, but hydrolases that remain active facilitate pathogen survival. We targeted Mtb esterases, a diverse set of enzymes in the serine hydrolase family, and studied their activities using both activity-based probes (ABPs) and fluorogenic esterase substrates. These small-molecule probes revealed functional esterases in active, dormant, and reactivating cultures. Using ABPs, we identified five esterases that remained active in dormant Mtb, including LipM (Rv2284), LipN (Rv2970c), CaeA (Rv2224c), Rv0183, and Rv1683. Three of these, CaeA, Rv0183, and Rv1683, were catalytically active in all three culture conditions. Fluorogenic probes additionally revealed LipH (Rv1399c), Culp1 (Rv1984c), and Rv3036c esterase activity in dormant and active cultures. Esterases with persistent activity are potential diagnostic biomarkers or therapeutic targets for Mtb-infected individuals with latent or active tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Esterasas/antagonistas & inhibidores , Tuberculosis Latente/microbiología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Antituberculosos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Esterasas/genética , Esterasas/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis/microbiologíaRESUMEN
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.
Asunto(s)
Esterasas/metabolismo , Mycobacterium tuberculosis/enzimología , Colorantes Fluorescentes/química , Resonancia Magnética Nuclear Biomolecular , Especificidad por SustratoRESUMEN
Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (Ï=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.