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Identification of bile acid-interacting proteins in Salmonella Typhimurium (14028S) by chemoproteomics

ABSTRACT: The microbiota generates structurally diverse small molecules that can regulate host physiology and disease. Of these microbiota metabolites, bile acids have emerged as important modulators of host immunity and microbial pathogenesis. While the modes of action for different bile acids on host pathways are becoming more apparent, the mechanisms by which these prominent microbiota metabolites suppress microbial virulence pathways are less clear. To identify the direct protein targets of bile acids in Salmonella, we have generated three photoaffinity bile acid reporters (alk-X-CDCA, alk-X-UDCA, alk-X-LCA) and performed chemical proteomics. Using a combination of photocrosslinking with bile acid chemical reporters and label-free proteomics, we performed a quantitative analysis of bile acid interacting proteins in Salmonella with or without UV-mediated photocrosslinking. These studies revealed bile acid can interact with many Salmonella proteins, such as extracellular or secreted proteins, T3SS components and motility-related proteins, as predicted by Gene Ontology analysis. In addition, cytoplasmic and membrane proteins including metabolic enzymes are also identified. Notably, HilD, an important transcriptional regulator of S. Typhimurium virulence was also identified by the alk-X-CDCA reporter. This study highlights the utility of chemical proteomics to identify the direct protein targets and mechanisms of action for microbiota metabolites in bacterial pathogens.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Typhimurium Str. Atcc 14028

SUBMITTER: Xinglin Yang

LAB HEAD: Howard C. Hang

PROVIDER: PXD034373 | Pride | 2022-09-29

REPOSITORIES: Pride

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			Action	DRS
	MS_Xinglin_Hang_DMSO-No-UV_1.raw	Raw
	MS_Xinglin_Hang_DMSO-No-UV_2.raw	Raw
	MS_Xinglin_Hang_DMSO-No-UV_3.raw	Raw
	MS_Xinglin_Hang_DMSO-No-UV_4.raw	Raw
	MS_Xinglin_Hang_alk-X-CDCA-No-UV_1.raw	Raw

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Project description:Infection with Salmonella enterica serovar Typhi in humans causes the systemic, life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. The ensuing disease is characterized by systemic dissemination and colonization of many organs, including the liver, spleen and gallbladder. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate. Additionally, we showed that Salmonella Typhimurium can use bile phospholipids to grow at high rates. These abilities are likely important for quick colonization of the gallbladder during typhoid fever and further pathogen dissemination through fecal shedding. To further characterize the interactions between Salmonella and the gallbladder environment we compared the transcriptome of Salmonella cultures grown in LB or physiological murine bile. Our data showed that many genes involved in bacterial central metabolism are affected by bile, with the citric acid cycle being repressed and alternative respiratory systems being activated. Additionally, our study revealed a new aspect of Salmonella interactions with bile through the identification of phoP as a bile-responsive gene. Repression of phoP expression does not involve PhoPQ sensing of a bile component. Due to its critical role in Salmonella virulence, further studies in this area will likely reveal aspects of the interaction between Salmonella and bile that are relevant to disease. Salmonella enterica serovar Typhimurium LT2 glass slide microarrays were obtained through the NIAID’s Pathogen Functional Genomics Resource Center, managed and funded by Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS and operated by the J. Craig Venter Institute. Two independent cultures of Salmonella enterica serovar Typhimurium SL1344 were grown overnight in Luria-Bertani (LB) broth with streptomycin (100 μg/mL) at 37oC with shaking (225 RPM). Cells were pelleted by centrifugation and resuspended in sterile Dulbecco’s phosphate-buffered saline (PBS). These suspensions were further diluted 1:50 in PBS and used to inoculate 70 μL of either LB or physiological murine bile (in 0.65-mL tubes) at a 1:100 dilution. Cultures were incubated for 24 hours at 37oC with agitation (225 RPM). Then, samples were diluted in PBS and serial dilutions were plated on LB plates containing 100 μg/mL of streptomycin and incubated overnight at 37oC for bacterial growth and enumeration by colony counting. The remaining cells were used in the subsequent steps, as described below. RNA isolation began with the addition of 2 volumes of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) to the bacterial cultures and incubation at room temperature for approximately 5 minutes. Cells were pelleted by centrifugation and RNA was isolated using the RNeasy Mini Kit (Qiagen) with the on-column DNase digestion, according to the manufacturer’s recommendations. Synthesis of cDNA was performed as described herein. The entire RNA sample (≥3 μg) was mixed with 6 μg of Random Primers (Invitrogen, Burlington, Canada) and 40 units of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen, Burlington, Canada) in a total volume of 18.5 μL and incubated at 70oC for 10 minutes. Samples were incubated on ice for 30 seconds, centrifuged to bring down condensation and incubated with the following components (final concentrations; Invitrogen): First Strand Buffer (1X), dithiothreitol (DTT; 10 mM), dNTPs (dATP, dCTP, dGTP, dTTP; 0.5 mM each) and 100 units of SuperScript II reverse transcriptase in a total volume of 30 μL. The mixture was incubated at 42oC overnight and the reaction was stopped and the RNA degraded through the addition of 0.5 M EDTA and 1 M sodium hydroxide (10 μL each) and incubation at 65oC for 15 minutes. Then, 25 μL of 1 M Tris (pH 7) was added to neutralize the pH of the cDNA solution. cDNA was purified using the MinElute PCR Purification Kit (Qiagen) according to the manufacturer’s recommendations, except that the Qiagen wash and elution buffers were substituted by phosphate buffers, according to the PFGRC microarray protocol. cDNA was then precipitated using ammonium acetate and ethanol and labeled with Cy3 (LB cultures) and Cy5 (bile cultures) using the ULS aRNA Fluorescent Labeling Kit (KREATECH Biotechnology, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Samples were mixed, dried and saved at -80oC until used. Pre-hybridization, hybridization and washing steps were performed essentially as described in the PFGRC protocols, except that the hybridization buffer contained KREAblock blocking buffer (25%; KREATECH Biotechnology). After hybridization, slides were scanned using an Affymetrix 428 Array Scanner from Eurofins MWG Operon (Huntsville, USA).

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Identification of bile acid-interacting proteins in Salmonella Typhimurium (14028S) by chemoproteomics

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