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Intracellular proliferation and virulence gene expression of Salmonella are influenced by pre-invasion growth conditions

ABSTRACT: Salmonella invasion of non-phagocytic cells is a dynamic process that requires the translocation of effector proteins into host cells by a type III secretion system (T3SS1) encoded on Salmonella pathogenicity island 1 (SPI1). The signals involved in T3SS1 induction in vivo are not known, but SPI1-induced invasive Salmonella can be prepared by growth in synthetic media. Here we compared two different methods of preparing SPI1-induced bacteria by using a combination of transcriptome analysis, a single-cell reporter assay and intracellular gene expression analysis to reveal clear differences between these two bacterial populations that are reflected in their ability to interact with cultured epithelial cells. Invasion efficiency and intracellular replication were greater for bacteria grown with aeration to late-log phase compared to those grown without aeration to stationary phase. No significant differences in SPI1 regulon expression were revealed by transcriptome analysis. By contrast, surface appendage and stress response genes were considerably changed. Single cell analyses of gene expression revealed variation in the frequency of SPI1-induced bacteria and a correlation between SPI1-induction and the presence of flagella. The expression of virulence genes following invasion of cultured epithelial cells was also found to be predetermined by pre-invasion growth conditions. Key words: epithelial, flagella, invasion, motility, Salmonella-containing vacuole,

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

PROVIDER: GSE13343 | GEO | 2010/04/19

SECONDARY ACCESSION(S): PRJNA109865

REPOSITORIES: GEO

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Project description:MicroRNAs have well-established roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. In contrast, microRNA responses to invasive bacteria have remained unknown. Here, we report cell type-dependent microRNA regulations upon infection of mammalian cells with the enteroinvasive pathogen, Salmonella Typhimurium. Murine macrophages strongly up-regulate NF-κB associated microRNAs; strikingly, these regulations which are induced by the bacterial lipopolysaccharides (LPS) occur and persist regardless of successful host invasion and/or replication, or whether an inflammatory response is mounted, suggesting that microRNAs belong to the first line of anti-bacterial defence. However, a suppression of the global immune regulator miR-155 in endotoxintolerant macrophages revealed that microRNA responses also depend on the status of infected cells. This study identifies the let-7 family as the common denominator of Salmonella regulated microRNAs in macrophages and epithelial cells, and suggests that repression of let-7 relieves cytokine IL-6 and IL-10 mRNAs from negative post-transcriptional control. Our results establish a paradigm of microRNA-mediated feed-forward activation of inflammatory factors when mammalian cells are targeted by bacterial pathogens. The murine macrophage cell-line RAW 264.7 was exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. We compared mouse mRNA abundances between cells infected with wild-type ("24WT") or SPI1/2 knockout Salmonella ("24SPI1/2") and non-infected, mock-treated controls ("24C"). Both strains mounted a similar inflammatory response as judged from the abundance of pro-inflammatory genes compared to the controls. These data indicate, that macrophages can mount a full inflammatory response to extracellular bacteria, that is not further enhanced or attenuated by the sensing of intracellular bacteria. Murine RAW 264.7 cells were rendered endotoxin-tolerant by pre-exposure to heat-killed Salmonella typhimurium SL1344 ("HKS") at an MOI of 10 for 20 h. 5 days upon removal of the HKS stimulus cells were exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. The wild-type Salmonella strain invades and replicates in endotoxin-tolerant RAW 264.7 cells, though the intracellular replication rate is significantly reduced (by roughly 40 %) compared to naive RAW 264.7 cells. We compared mouse mRNA abundances between cells infected with wild-type ("wt") and non-infected, mock-treated controls ("Mock") or between wild-type ("wt") and SPI1/2-knockout ("SPI1/2") infected cells. As apparent from the Microarray data the wild-type Salmonella strain induces a typical inflamatory response in endotoxin-tolerant RAW 264.7 cells (as judged from the fold-induction of NFkB-inducible genes). As expected however, mRNAs corresponding to known inflammatory genes are significantly less abundant in response to the non-invasive SPI1/2 knockout strain, compared to wild-type infection. These data indicate, that endotoxin-tolerance renders macrophages largely insensitive to extracellular, but not to intracellular bacteria. ***This submission represents the mRNA component of the study

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Intracellular proliferation and virulence gene expression of Salmonella are influenced by pre-invasion growth conditions

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