Identification of common highly expressed genes of Salmonella Enteritidis by in silico prediction of gene expression and in vitro transcriptomic analysis
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ABSTRACT: We report the use of RNA-Seq technique to identify common highly expressed genes in pathogenic poultry-associated field strains of S. Enteritidis grown in LB-salt broth (300mM NaCl) and low-phosphate/low -magnesium/low-pH LPM broth at avian body temperature (42°C).
Project description:We report the use of RNA-Seq technique to characterize differences in the global transcritome between multiple genetically similar but phenotypically diverse poultry-associated field strains of S. Enteritidis grown in laboratory media at avian body temperature (42°C).
Project description:comparison of total RNA profiles obtained from ciprofloxacin resistant colonies of two separate Enteritidis isolates with their respective wt parents total RNA of both wt (with reduced cip sensitivity at MIC=0.2 ug/ml) and spontaneous cipR mutants (MIC>32ug/ml) in LB log phase was harvested from Enteritidis strain 104 (from poultry host) and Enteritidis 5408 (from human host), and compared on a PCR product array
Project description:Investigation of whole genome gene expression level changes in Salmonella enterica serova Enteritidis and Typhimurium under chlorine treatment An eighteen chip study using total RNA isolated from three separate cultures of (1) S. Enteritidis in BHI broth (2) S. Typhimurium in BHI broth (3) S. Enteritidis in BHI broth w/ 130 ppm chlorine (4) S. Typhimurium in BHI w/ 130 ppm chlorine (5) S. Enteritidis in BHI broth w/ 390 ppm (6) S. Typhimurium in BHI broth w/ 390 ppm. Each chip measures the expression level of 5,027 ORFs covering the whole genome of S. Enteritidis and S. Typhimurium.
Project description:S. aureus strain JKD6009 RNase III-HTF and USA300 RNase III-HTF were used in our study to capture the RNA-binding proteome in Staphylococcus aureus. Both strains were inoculated in TSB (Tryptone soya broth, Oxioid CM0129) and overnight cultured in 37°C, 200 rpm shaker. Strain JKD6009 RNase III-HTF was sub-cultured into 190 ml LPM (Coombes et al., 2004) (Low phosphate, low magnesium medium, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 8 µM MgCl2, 1 M KH2PO4, 16 mM Tris-HCl, 0.1% casamino acids, 0.3% Glycerol) and grew from OD600 0.01 to 1. Overnight cultures of USA300 RNase III-HTF was re-inoculated to 65 ml fresh TSB to OD600 0.05 and left to grow to an OD600 of ~3. Cells were then filtered through 0.45 µm filters using a vacuum filtration device (UVO3; (McKellar et al., JoVe 2020; Van Nues et al., Nature Communications 2017)) shifted to same volume of LPM medium for 15 min at 37ºC and UV irradiated in LPM in the Vari-X-linker (λ =254 nm) (https://www.vari-x-link.com; (McKellar et al., 2020; Van Nues et al., 2017)) with an energy dose of 2J/cm2. In total, 6 replicates were collected (three technical and two biological replicates) for each experiment.
Project description:S. aureus strain JKD6009 RNase III-HTF and USA300 RNase III-HTF were used in our study to capture the RNA-binding proteome in Staphylococcus aureus. Both strains were inoculated in TSB (Tryptone soya broth, Oxioid CM0129) and overnight cultured in 37°C, 200 rpm shaker. Strain JKD6009 RNase III-HTF was sub-cultured into 190 ml LPM (Coombes et al., 2004) (Low phosphate, low magnesium medium, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 8 µM MgCl2, 1 M KH2PO4, 16 mM Tris-HCl, 0.1% casamino acids, 0.3% Glycerol) and grew from OD600 0.01 to 1. Overnight cultures of USA300 RNase III-HTF was re-inoculated to 65 ml fresh TSB to OD600 0.05 and left to grow to an OD600 of ~3. Cells were then filtered through 0.45 µm filters using a vacuum filtration device (UVO3; (McKellar et al., JoVe 2020; Van Nues et al., Nature Communications 2017)) shifted to same volume of LPM medium for 15 min at 37ºC and UV irradiated in LPM in the Vari-X-linker (λ =254 nm) (https://www.vari-x-link.com; (McKellar et al., 2020; Van Nues et al., 2017)) with an energy dose of 2J/cm2. In total, 6 replicates were collected (three technical and two biological replicates) for each experiment.
Project description:S. aureus strain JKD6009 RNase III-HTF and USA300 RNase III-HTF were used in our study to capture the RNA-binding proteome in Staphylococcus aureus. Both strains were inoculated in TSB (Tryptone soya broth, Oxioid CM0129) and overnight cultured in 37°C, 200 rpm shaker. Strain JKD6009 RNase III-HTF was sub-cultured into 190 ml LPM (Coombes et al., 2004) (Low phosphate, low magnesium medium, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 8 µM MgCl2, 1 M KH2PO4, 16 mM Tris-HCl, 0.1% casamino acids, 0.3% Glycerol) and grew from OD600 0.01 to 1. Overnight cultures of USA300 RNase III-HTF was re-inoculated to 65 ml fresh TSB to OD600 0.05 and left to grow to an OD600 of ~3. Cells were then filtered through 0.45 µm filters using a vacuum filtration device (UVO3; (McKellar et al., JoVe 2020; Van Nues et al., Nature Communications 2017)) shifted to same volume of LPM medium for 15 min at 37ºC and UV irradiated in LPM in the Vari-X-linker (λ =254 nm) (https://www.vari-x-link.com; (McKellar et al., 2020; Van Nues et al., 2017)) with an energy dose of 2J/cm2. In total, 6 replicates were collected (three technical and two biological replicates) for each experiment.
Project description:Global gene expression profiling in spleen in chicken inoculated with S. enteritidis and control was conducted to screen differentially expressed genes and identify the key gene, signalling pathways and important biological processes that related to the infection of S. enteritidis
Project description:Comparative genomic hybridization of a temporally and locally diverse set of S. enterica ssp I serovar Enteritidis isolates, and some closely related serovar Dublin and Gallinarum strains, to the sequenced Enteritidis PT4 Keywords: other
Project description:Salmonella Enteritidis is the major food-borne pathogen primarily causing human infection through contaminated chicken meat and eggs. We recently demonstrated that S. Enteritidis strains from poultry differ in their ability to invade human intestinal cells and cause disease in orally challenged mice. Here we hypothesized that the differential pathogenicity of S. Enteritidis strains is due to the differential fitness in the adverse environments that may be encountered during infection in the host. The response of a panel of six S. Enteritidis strains to acid stress, oxidative stress, survival in egg albumen and the ability to cause infection in chickens were analyzed. This analysis allowed classification of strains into two categories: stress- sensitive and stress- resistant, with the former showing significantly (P<0.05) reduced survival in acidic (gastric phase of infection) and oxidative (intestinal and systemic phase of infection) stress. Stress-sensitive strains also showed impaired intestinal colonization and systemic dissemination in orally inoculated chickens and failed to survive/grow in egg albumen. Comparative genomic hybridization microarray analysis revealed no differences at the discriminatory level of the whole gene content between stress-sensitive and stress-resistant strains. However, sequencing of rpoS, a stress-regulatory gene, revealed that one of the three stress-sensitive strains carried an insertion mutation in the rpoS resulting in truncation of σS. Finding that one of the stress-sensitive strains carried an easily identifiable small polymorphism within a stress-response gene suggests that the other strains may also have small polymorphisms elsewhere in the genome, which likely impact regulation of stress or virulence associated genes in some manner.
Project description:Even though the incidence of salmonellosis in humans has decreased over the last years, Salmonella spp. are still a leading cause of foodborne outbreaks in Europe (Anon., 2014). Of more than 2500 different serovars of Salmonella enterica, S. enterica serovar Enteritidis (S. Enteritidis) is the most frequently reported serovar in relation to food borne disease, and egg and egg products are the most important vehicles (Anon., 2014). It has recently been shown that S. Enteritidis is superior to other serovars tested regarding survival in egg white, which may explain why many egg borne outbreaks are caused by this serovar (De Vylder et al., 2013). The genetic background for this apparent better adaptation to survival in egg is only partially known. The aim of this work was to carry out gene expression analysis in order to understand how S. Enteritidis adapts to growth in the hostile environment of egg. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using an improved modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acids biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg.