Project PXD011041

PRIDE Assigned Tags:
Biological Dataset Biomedical Dataset

Summary

Title

A multi-condition inter-strain transcriptomic compendium identifies phenotypic differences between European and African Salmonella pathovariants (C4PR_LIV)

Description

Salmonella Typhimurium ST313 is associated with invasive nontyphoidal salmonellosis (iNTS) disease in sub-Saharan Africa. Using ST313 isolate, D23580, and the well-characterized ST19 isolate 4/74, that causes gastroenteritis across Europe, we generated transcriptomic data from sixteen infection-relevant in vitro conditions and during infection of murine macrophages (http://bioinf.gen.tcd.ie/cgi-bin/jay/salcom_v2.pl?db=salcom_africa_new_HL). The transcriptional response of the two S. Typhimurium pathovariants to environmental stress was broadly similar. Comparative gene expression analysis identified virulence and metabolic genes that were up/down-regulated in D23580 versus 4/74. Findings were backed up by proteomic analysis, with12 genes being differentially-expressed at both the transcriptional and protein levels including melA and cysS. The data led us to discover that the ablation of melibiose utilization was caused by 3 SNP mutations in D23580 and to identify a novel plasmid-maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase. Our functional transcriptomic analysis represents a powerful tool for identification of key phenotypic differences between bacterial pathovariants.

Sample Processing Protocol

A LC-MS/MS (Q Exactive orbitrap) 4 h reversed phase C18 gradient was used to generate proteomic data from six biological replicates of each strain, 4/74 and D23580, grown in the ESP condition in Lennox broth. Pellet from bacterial cultures was resuspended in 50 mM phosphate buffer (pH 8), sonicated (10 sec ON, 50 sec OFF, for 10 cycles at 30% amplitude), and supernatants were analyzed after centrifugation at 16,000 g for 20 min. Subsequent experimental procedures were performed at the Centre for Proteome Research at the University of Liverpool. In brief, 100 μg of protein were digested (RapiGest, in-solution trypsin digestion) and 1 μg of digested protein was run on a LC-MS/MS platform.

Data Processing Protocol

A database was generated merging the amino acid sequences of the annotated genes in 4/74 [27] and our re-annotated D23580 to allow homologous proteins as well as strain-specific proteins to be identified. The merged database was clustered using the program Cd-hit and an identity threshold of 95% [84]. Clusters with a single protein, representing strain-specific proteins, were included in the database with their accession ID. Clusters with more than one protein represented orthologues, and only peptides common to all proteins of the cluster were included in the database. Common peptides allowed label-free comparison of proteins that had a low level of sequence variation. Raw data obtained from the LC-MS/MS platform were loaded into the Progenesis QI software (Nonlinear Dynamics) for label-free quantification analysis. Differential expression analysis between the two strains, 4/74 and D23580, is shown in Table S6. From those results (2,013 proteins), multihit proteins (peptides assigned to more than one protein in the same strain) were removed leaving a total of 2,004 proteins. Cut-offs of ≥ 2 unique peptides per identified protein (1,632 proteins), ≥ 2 fold-change expression and p-value < 0.05 between strains (121 proteins) were used. Among the 121 proteins, 25 were 4/74-specific, 30 were D23580-specific, and 66 were encoded by orthologous genes between strains.

Contact

Philip Brownridge, University of Liverpool
Jay C. D. Hinton, Professor of Microbial Pathogenesis Institute of Integrative Biology Room 308, Biosciences Building, University of Liverpool Crown Street, Liverpool L69 7ZB, United Kingdom ( lab head )

Submission Date

10/09/2018

Publication Date

11/01/2019

Instrument

Q Exactive

Software

Not available

Experiment Type

Shotgun proteomics

Publication

    Canals R, Hammarlöf DL, Kröger C, Owen SV, Fong WY, Lacharme-Lora L, Zhu X, Wenner N, Carden SE, Honeycutt J, Monack DM, Kingsley RA, Brownridge P, Chaudhuri RR, Rowe WPM, Predeus AV, Hokamp K, Gordon MA, Hinton JCD. Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580. PLoS Biol. 2019 17(1):e3000059 PubMed: 30645593