The development of new treatment options for bacterial infections requires access to new targets for antibiotics and antivirulence strategies. Chemoproteomic approaches are powerful tools for... Show moreThe development of new treatment options for bacterial infections requires access to new targets for antibiotics and antivirulence strategies. Chemoproteomic approaches are powerful tools for profiling and identifying novel druggable target candidates, but their functions often remain uncharacterized. Previously, we used activity-based protein profiling in the opportunistic pathogen Staphylococcus aureus to identify active serine hydrolases termed fluorophosphonate-binding hydrolases (Fph). Here, we provide the first characterization of S. aureus FphH, a conserved, putative carboxylesterase (referred to as yvaK in Bacillus subtilis) at the molecular and cellular level. First, phenotypic characterization of fphH-deficient transposon mutants revealed phenotypes during growth under nutrient deprivation, biofilm formation, and intracellular survival. Biochemical and structural investigations revealed that FphH acts as an esterase and lipase based on a fold well suited to act on a small to long hydrophobic unbranched lipid group within its substrate and can be inhibited by active site-targeting oxadiazoles. Prompted by a previous observation that fphH expression was upregulated in response to fusidic acid, we found that FphH can deacetylate this ribosome-targeting antibiotic, but the lack of FphH function did not infer major changes in antibiotic susceptibility. In conclusion, our results indicate a functional role of this hydrolase in S. aureus stress responses, and hypothetical functions connecting FphH with components of the ribosome rescue system that are conserved in the same gene cluster across Bacillales are discussed. Our atomic characterization of FphH will facilitate the development of specific FphH inhibitors and probes to elucidate its physiological role and validity as a drug target. Show less
Activity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity... Show moreActivity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity-based probes to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug of 5-fluorouracil and also has antimicrobial and anti-biofilm activity. Carmofur probes were originally designed to target human acid ceramidase, a member of the NTN hydrolase family with an active-site cysteine nucleophile. Here, we first profiled the targets of a fluorescent carmofur probe in live S. aureus under biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatography/mass spectrometry. Treatment with a carmofur-biotin probe led to enrichment of 20 enzymes from diverse families awaiting further characterization, including the NTN hydrolase-related IMP cyclohydrolase PurH. However, the probe preferentially labeled serine hydrolases, thus displaying a reactivity profile similar to that of carbamates. Our results suggest that the electrophilic N-carbamoyl-5-fluorouracil scaffold could potentially be optimized to achieve selectivity towards diverse enzyme families. The observed promiscuous reactivity profile suggests that the clinical use of carmofur presumably leads to inactivation of a number human and microbial enzymes, which could lead to side effects and/or contribute to therapeutic efficacy. Show less
