Dataset Information

Comparative analysis of Salmonella Typhi, Paratyphi A, Enteritidis and Typhimurium core proteomes

ABSTRACT: Salmonella enterica subsp. enterica contains more than 2,600 serovars of which four are of major medical relevance for humans. While the typhoidal serovars (Typhi and Paratyphi A) are human-restricted and cause enteric fever, non-typhoidal Salmonella serovars (Typhimurium and Enteritidis) have a broad host range and predominantly cause gastroenteritis. In this study, we compared the core proteomes of Salmonella Typhi, Paratyphi A, Typhimurium and Enteritidis using contemporary proteomics. Five isolates, covering different geographical origins, and one reference strain per serovar were grown in vitro to the exponential phase. Protein levels of orthologous proteins between serovars were compared and subjected to gene ontology term enrichment and inferred regulatory interactions. Differential expression of the core proteomes of the typhoidal serovars appears mainly related to cell surface components and, for the non-typhoidal serovars, to pathogenicity. Our findings may guide future development of novel diagnostics and vaccines, and understanding of disease progression.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Paratyphi A Str. Atcc 9150 Salmonella Enterica Subsp. Enterica Serovar Typhi Str. Ct18 Salmonella Typhimurium (strain 14028s / Sgsc 2262) Salmonella Enterica Subsp. Enterica Serovar Enteritidis Str. P125109

TISSUE(S): Cell Culture

SUBMITTER: Sara Saleh

LAB HEAD: Stijn Deborggraeve

PROVIDER: PXD011154 | Pride | 2019-05-13

REPOSITORIES: Pride

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Publications

Salmonella Typhi, Paratyphi A, Enteritidis and Typhimurium core proteomes reveal differentially expressed proteins linked to the cell surface and pathogenicity.

Saleh Sara S Van Puyvelde Sandra S Staes An A Timmerman Evy E Barbé Barbara B Jacobs Jan J Gevaert Kris K Deborggraeve Stijn S

PLoS neglected tropical diseases 20190524 5

<h4>Background</h4>Salmonella enterica subsp. enterica contains more than 2,600 serovars of which four are of major medical relevance for humans. While the typhoidal serovars (Typhi and Paratyphi A) are human-restricted and cause enteric fever, non-typhoidal Salmonella serovars (Typhimurium and Enteritidis) have a broad host range and predominantly cause gastroenteritis.<h4>Methodology/principle findings</h4>We compared the core proteomes of Salmonella Typhi, Paratyphi A, Typhimurium and Enterit ...[more]

PMID: 31125353

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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).

Project description:During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to successfully occur. This requires bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Besides being resistant to it, Salmonella can also successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. In order to gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit. For in vitro studies, bile was extracted from C57BL/6 mice and used immediately. An overnight culture of Salmonella enterica serovar Typhimurium SL1344 was used to inoculate 10 ?L of bile at an approximate density of 5x10^6 cells/mL. The experiment was performed in duplicate, and a total of 2 uninfected and 2 infected bile samples were studied. Samples were incubated for 24 hours at 37 oC with shaking. After incubation, samples were centrifuged to remove bacteria and the supernatant was saved for metabolomic analyses. For in vivo studies, C57BL/6 mice were infected with approximately 10^8 bacterial cells by oral gavage. Four groups of three to four mice each were either infected with Salmonella or kept uninfected (total of 11 mice per treatment group). Five days after infection, all mice were sacrificed and bile was collected. Samples were centrifuged and supernatants saved for metabolomic analyses. Samples were prepared by mixing equal volumes of bile from three to four mice, generating three samples per treatment (uninfected and infected), which were used in the subsequent steps. Seven microliters of each sample was evaporated and the residue resuspended in 50% acetonitrine. Extracts were infused into a 12-T Apex-Qe hybrid quadrupole-FT-ICR mass spectrometer equipped with an Apollo II electrospray ionization source, a quadrupole mass filter and a hexapole collision cell. Raw mass spectrometry data were processed as described elsewhere (Han et al. 2008. Metabolomics. 4:128-140). To identify differences in metabolite composition between different groups of samples, we filtered the list of masses for metabolites which were present on one set of samples but not the other. Additionally, we calculated the ratios between averaged intensities of metabolites from each group of mice. To assign possible metabolite identities to the masses selected as described above, the monoisotopic neutral masses of interest were queried against the Human Metabolome Database (HMDB, http://www.hmdb.ca), with a tolerance of 0.001 Da.

Dataset Information

Comparative analysis of Salmonella Typhi, Paratyphi A, Enteritidis and Typhimurium core proteomes

Publications

Salmonella Typhi, Paratyphi A, Enteritidis and Typhimurium core proteomes reveal differentially expressed proteins linked to the cell surface and pathogenicity.

Similar Datasets

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