Project description:Many non-typhoidal serovars of Salmonella such as Salmonella enterica serovar Typhimurium (S. Typhimurium) are the leading cause of food-borne gastroenteritis, resulting in millions of infections each year and sometimes death. Salmonella enterica serovar Typhimurium is the most common non-typhoidal Salmonella strain isolated from patients around the world and is used as a mouse model to study bacterial pathogenesis and host-microbe interactions. Furthermore, S. Typhimurium is an important pathogen in livestock animals including chickens and cattle. S. Typhimurium utilises a multitude of virulence factors to reach and invade host cells and for its intracellular survival. However, little is known about the mechanism of protein synthesis of these virulence factors at the codon level. Here, we performed RNA-seq and ribosome profiling. Ribosome profiling allows the global mapping of translating ribosomes on the transcriptome and therefore provides direct measure of protein synthesis.
Project description:1-day-old C57BL/6 mice were left untreated (co) or orally infected with 10E2 CFU wildtype (wt) or delta invC SPI1 mutant Salmonella Typhimurium (ATCC14028). Four biological replicates obtained from individual animals were exmained; each group contained animals from at least 2 different litters. On day 4 p.i., animals were sacrificed and intestinal epithelial cells were isolated from total small intestine (protocol according to: Lotz et al., J. Exp. Med. 2006). Total RNA was isolated by TriZol and its purity was examined using a Bioanalyzer. We used microarrays to detail the global gene expression in primary total isolated intestinal epithelial cells.
Project description:Salmonella enterica serovar Agona (S. Agona) is a foodborne pathogen that caused recurrent multistate outbreaks associated with cereal between 1998 and 2008, underscoring the endurance of Salmonella over time in low-moisture food (LMF) processing facilities. In this study, we aimed to determine the molecular mechanism of survival of S. Agona in LMF and confirm their impact on phenotype by the knockout study. S. Agona strain (CFSAN 000477), isolated from cereal, was selected for this study. A 100µl suspension with a concentration of ~10^11 cfu/ml was inoculated into 3g of rice cereals. Three replications of inoculated cereals were subjected to desiccation stress (aw ≤ 0.25) for 24h at room temperature (25⁰C). Inoculated cereal samples were collected at 6 timepoints post-inoculation. Cells were separated from the food matrix for RNA extraction. RNA sequencing was performed using the NextSeq 2000 platform. Read counts were generated with Salmon v1.9.0. Downstream analysis was conducted with R and KEGG mapper. There were 1120 differentially expressed genes (DEGs) in S. Agona in response to desiccation stress (Padj < 0.01, |log2FoldChange| >1), with 647 downregulated and 473 upregulated. Functional analysis of downregulated DEGs revealed that most of the genes were associated with metabolic pathways, followed by translation, suggesting slower growth in the surviving population. The top 3 upregulated genes/operons: kdp and ccm operon, and tisB were knocked out and checked for survival study. Approximately 1-2 log reduction (p>0.05) was noticed in the survival of the mutants compared with the wild type. This transcriptome data suggests that Salmonella Agona survives in low-moisture food by conserving energy, lowering metabolism, and reducing replication.
