Chemoproteomic Profiling Reveals the Mechanism of Bile Acid Tolerance in Bacteria

Abstract

Bile acids (BAs) are a class of endogenous metabolites with important functions. As amphipathic molecules, BAs have strong antibacterial effects, preventing overgrowth of the gut microbiota and defending the invasion of pathogens. However, some disease-causing pathogens can survive the BA stress and knowledge is limited about how they develop BA tolerance. In this work, we applied a quantitative chemoproteomic strategy to profile BA-interacting proteins in bacteria, aiming to discover the sensing pathway of BAs. Using a clickable and photo-affinity BA probe with quantitative mass spectrometry, we identified a list of histidine kinases (HKs) of the two-component systems (TCS) in bacteria as the novel binding targets of BA. Genetic screening revealed that knocking out one specific HK, EnvZ, renders bacteria with significant sensitivity to BA. Further biochemical and genetic experiments demonstrated that BA binds to a specific pocket in EnvZ and activates a downstream signaling pathway to help efflux of BA from bacteria, resulting in BA tolerance. Collectively, our data revealed that EnvZ is a novel sensor of BA in bacteria and its associated TCS signaling pathway plays a critical role in mediating bacterial BA tolerance, which opens new opportunities to combat BA-tolerating pathogens.

Chemical proteomic analysis of bile acid-protein targets in Enterococcus faecium

“…Next, we performed enzyme kinetics analysis of the deconjugation reaction. TLCA of increasing concentrations (1,5,10,20,50,100,200, 500 mM) were incubated with 100 nM BSH for 1 min. The input of TLCA and production of LCA were examined by LC-MS (Fig.…”

“…18 Of note, photoaffinity reporters were developed for primary bile acid cholic acid and enabled the identification of cholic acidbinding proteins in mammalian cell lines 19 and E. coli. 20 In addition, our chemoproteomic analysis in Salmonella revealed anti-infective bile acids such as chenodeoxycholic acid can bind and inactivate a transcriptional regulator of Salmonella virulence HilD. 21 Furthermore, chemical proteomic analysis of bile acid targets in Clostridium difficile showed that lithocholic acid (LCA) can bind and modulate the function of a stress response transcriptional regulator BapR.…”

“…Alternatively, photoaffinity reporters for bile acids and other microbiota metabolites can be employed to characterize other protein targets in mammalian cells and microbes 18 . Of note, photoaffinity reporters were developed for primary bile acid cholic acid and enabled the identification of cholic acid-binding proteins in mammalian cell lines 19 and E. coli 20 . In addition, our chemoproteomics analysis in Salmonella revealed anti-infective bile acids such as chenodeoxycholic acid can bind and inactivate a transcriptional regulator of Salmonella virulence HilD 21 .…”

Bifunctional Lipid-Derived Affinity-Based Probes (AfBPs) for Analysis of Lipid–Protein Interactome

“…Among the identified proteins, 265 of them, including some known cholesterol-binding proteins and processing enzymes, were shown to be sensitive to cholesterol competition and selective for trans -sterol over the nonsteroidal neutral lipid probe ( N -palmitoylethanolamine-diazirine-alkyne). The Lei and Wang groups jointly reported a bile acid (BA) based A f BP (CA) with an alkyne and a diazirine installed at the C24 and C3 positions, respectively (Figure G). , Over 600 BA-interacting protein targets were subsequently identified in mammalian proteomes . Furthermore, a histidine kinase (EnvZ) was revealed to cause BA tolerance in bacteria …”

“…The Lei and Wang groups jointly reported a bile acid (BA) based A f BP (CA) with an alkyne and a diazirine installed at the C24 and C3 positions, respectively (Figure G). , Over 600 BA-interacting protein targets were subsequently identified in mammalian proteomes . Furthermore, a histidine kinase (EnvZ) was revealed to cause BA tolerance in bacteria …”