Proteomic Characterization of Virulence Factors and Related Proteins in Enterococcus strains from Dairy and Fermented Food Products
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap
ORGANISM(S): Enterococcus Faecalis (streptococcus Faecalis)
SUBMITTER: 
Mónica Carrera
LAB HEAD: Monica Carrera
PROVIDER: PXD036435 | Pride | 2022-10-13
REPOSITORIES: Pride
Publications
Proteomic Characterization of Virulence Factors and Related Proteins in <i>Enterococcus</i> Strains from Dairy and Fermented Food Products.
Abril Ana G AG Quintela-Baluja Marcos M Villa Tomás G TG Calo-Mata Pilar P Barros-Velázquez Jorge J Carrera Mónica M
International journal of molecular sciences 20220919 18
Enterococcus species are Gram-positive bacteria that are normal gastrointestinal tract inhabitants that play a beneficial role in the dairy and meat industry. However, Enterococcus species are also the causative agents of health care-associated infections that can be found in dairy and fermented food products. Enterococcal infections are led by strains of Enterococcus faecalis and Enterococcus faecium, which are often resistant to antibiotics and biofilm formation. Enterococci virulence factors ...[more]
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Project description:Inappropriate response to normal intestinal bacteria is involved in the development of Inflammatory Bowel Diseases (IBD, e.g. Crohnâ??s Disease (CD), ulcerative colitis (UC)) and variations in the host genome may mediate this process. IL-10-/- mice develop CD-like colitis mainly in the colon, in part due to inappropriate responses to normal intestinal bacteria including Enterococcus strains. Comprehensive characterization of changes in gene expression associated with the observed inflammation in the IL-10-/- mouse model has yet to be reported. Our aim was to characterize changes in colonic gene expression in IL-10-/- and C57BL/6J (C57; control) mice resulting from oral bacterial inoculation with 12 Enterococcus faecalis and faecium (EF) strains isolated from calves or poultry, complex intestinal flora (CIF) collected from healthy control mice, or a mixture of the two (EFâ?¢CIF). At 12 weeks of age, total RNA extracted from intact colon was hybridized to Agilent 44k mouse arrays. Lists of differentially expressed genes were generated using linear models for microarray analysis (Bioconductor), and genes clustered using GeneSpring GX and Ingenuity Pathways Analysis software. Intestinal inflammation was increased in IL-10-/- mice as a result of inoculation, with the strongest effect being in the EF and EF.CIF groups; expression of >4,000 gene probes was altered in colon tissue compared with similarly inoculated C57 mice. Genes differentially expressed in IL-10-/- mice as a result of EF or EF.CIF inoculation were associated with the following pathways: inflammatory disease (111 genes differentially expressed), immune response (209 genes), antigen presentation (11 genes, particularly major histocompatability complex Class II), fatty acid metabolism (30 genes) and detoxification (31 genes). Our results suggest that the colonic inflammation in EF and EF.CIF-inoculated IL-10-/- mice is mediated by mechanisms similar to those of human IBD, in particular CD. Example logFC calculation; This example describes the calculation of the fold-change value (logFC) for a single spot (in this case, spot 27222) for a single comparison. This calculation method was applied to generate all of the comparison data shown in the supplementary data files. The comparison in question is for:; colon tissue from C57BL/6J mouse with EF.CIF treatment (represented by duplicate samples GSM307282 (Title: C57BL/6J_Colon_EF.CIF_Rep1_(Pool17)) and GSM307283 (Title: C57BL/6J_Colon_EF.CIF_Rep2_(Pool18)); vs. colon tissue from C57BL/6J mouse under conventional conditions (represented by duplicate samples GSM307270 (Title: C57BL/6J_Colon_Conventional_Rep1_(Pool5)) and GSM307271 (Title: C57BL/6J_Colon_Conventional_Rep2_(Pool6)); Important Note: Limma analysis maps the treatments on each slide to the M values which are by convention always log2(R) - log2(G). Because this experiment is a reference design, with the reference RNA always Cy5 (i.e. red, channel 2) and the treatment sample Cy3 (i.e. green, channel 1), and we are interested in the values of the treatment samples compared with the reference, the M values in the data files are displayed as log2(G) - log2(R); The average expression is what is important here, which is calculated from the normalized M values for the appropriate comparison of interest as follows:; [1] Mean value for Spot 27222, for each treatment:; C57BL/6J mouse, EF.CIF: = mean (0.26363587 (GSM307282), 0.05084283 (GSM307283)) = 0.15723935 C57BL/6J mouse, conventional = mean (1.42951674 (GSM307270),1.15444106 (GSM307271)) = 1.2919789; [2] Calculation of LogFC for the comparison of interest:; The contrast of interest is C57BL/6J: EF.CIF vs Conventional, so by performing a subtraction of these values for the comparison of interest (i.e. (mean for EF.CIF) - (mean for Conventional), the common reference is cancelled out, thus:; C57BL/6J: EF.CIF vs Conventional = 0.15723935 - 1.2919789 = -1.13473955; So, the fold-change value (logFC) for spot 27222 for the comparison in question (C57BL/6J: EF.CIF vs. Conventional) is: -1.13473955 Experiment Overall Design: Animals. Twenty five male IL-10-/- (C57BL/6J background, formal designation B6.129P2-IL-10<tm1Cgn>/J) mice and twenty five male C57BL/6J (C57) control mice were received from The Jackson Laboratory (Bar Harbor, Maine, USA) at approximately five weeks of age. Mice were housed either in pairs or groups of three (5 mice per treatment) in shoebox-style cages containing untreated wood shavings and a plastic tube for environmental enrichment. The animal room was maintained at a temperature of ~22°C and humidity of ~50% with a 12 hour light/dark cycle. All mice had ad libitum access to water, which was refreshed twice a week. An AIN-76A powdered diet was supplied twice a week, with sufficient provided to meet the daily intake of IL-10-/- mice, as determined in a previous feeding trial (data not shown). The diet for all groups was sterilized by gamma irradiation (Schering-Plough, Wellington, New Zealand) to a level required for SPF conditions, to minimize the possibility of bacterial introduction to the SPF group of animals. All mice were weighed three times weekly and carefully monitored for disease symptoms (weight loss, soft faeces and inactivity). Experiment Overall Design: Experimental design. Both IL-10-/- and C57 mice were randomly divided into five treatment groups with five animals per group. Experiment Overall Design: SPF. Housed in Specific Pathogen Free (SPF) conditions (isolator cages supplied with HEPA filtered air (Tecniplast SpA, Buguggiate, Italy)). Experiment Overall Design: C. Maintained under conventional conditions. Experiment Overall Design: EF. Maintained under conventional conditions and orally inoculated (200 microlitres) with a solutions containing 12 pure strains of Enterococcus (1.2 x 10^8 colony forming units (CFU)). Experiment Overall Design: CIF. 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