Biochemical and Cellular Characterization of the Function of Fluorophosphonate-Binding Hydrolase H (FphH) in Staphylococcus aureus Support a Role in Bacterial Stress Response

Abstract

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 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 f phH-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.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections

“…This structure also confirms the proposed reaction mechanism between compound 9 and FphE (Figure 3B). 42,56,57,65,68,69 The electron density clearly shows the covalent bond between the terminal oxygen atom of active site Ser103 and the opened oxadiazolone ring (Figure 3C). The two oxygen atoms of the opened ring are stabilized in the structure by hydrogen bonds to the nitrogen ring atom of His257 and the backbone nitrogen atom of Ala28 (Figure 3C,D).…”

“…Among the Fphs, we found that the lipid esterase FphB is likely a virulence factor important for infection in the heart and liver in vivo . In addition, recent structural and biochemical studies of FphF and FphH have begun to shed light on possible functions of these enzymes. , The importance of FphB and FphH in mediating systemic S. aureus infections makes them potential targets for development of therapeutic agents. , In order to develop therapeutic and diagnostic agents for Staphylococcus infections, it is optimal to select targets that are unique to this organism over other highly related bacterial species .…”

“…In addition, recent structural and biochemical studies of FphF and FphH have begun to shed light on possible functions of these enzymes. , The importance of FphB and FphH in mediating systemic S. aureus infections makes them potential targets for development of therapeutic agents. , In order to develop therapeutic and diagnostic agents for Staphylococcus infections, it is optimal to select targets that are unique to this organism over other highly related bacterial species . In particular, FphE fits this criterion as it is the least conserved Fph protein across 50 species within the Staphylococcus genus. , …”

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection ofS. aureusInfections

“…26 In addition, recent structural and biochemical studies of FphF and FphH have begun to shed light on possible functions of these enzymes. 27, 28 The importance of FphB and FphH in mediating systemic S. aureus infections, makes them potential targets for development of therapeutic agents. 26, 28 In order to develop therapeutic and diagnostic agents for Staphylococcus infections, it is optimal to select targets that are unique to this organism over other highly related bacterial species.…”

“…27, 28 The importance of FphB and FphH in mediating systemic S. aureus infections, makes them potential targets for development of therapeutic agents. 26, 28 In order to develop therapeutic and diagnostic agents for Staphylococcus infections, it is optimal to select targets that are unique to this organism over other highly related bacterial species. 29 In particular, FphE fits this criterion as it is the least conserved Fph protein across 50 species within the Staphylococcus genus.…”