1887

Abstract

Toll-like receptor 4 (TLR4) senses bacterial LPS and is required for the control of systemic serovar Typhimurium infection in mice. The mechanisms of TLR4 activation and its downstream signalling cascades are well described, yet the direct effects on the pathogen of signalling via this receptor remain unknown. To investigate this we used microarray-based transcriptome profiling of intracellular . Typhimurium during infection of primary bone marrow-derived macrophages from wild-type and TLR4-deficient mice. We identified 17 Typhimurium genes that were upregulated in the presence of functional TLR4. Nine of these genes have putative functions in oxidative stress resistance. We therefore examined . Typhimurium gene expression during infection of NADPH oxidase-deficient macrophages, which lack normal oxidative killing mechanisms. We identified significant overlap between the ‘TLR4-responsive’ and ‘NADPH oxidase-responsive’ genes. This is new evidence for a link between TLR4 signalling and NADPH oxidase activity. Interestingly, with the exception of a mutant, Typhimurium strains lacking individual TLR4- and/or oxidative stress-responsive genes were not attenuated during intravenous murine infections. Our study shows that TLR4 activity, either directly or indirectly, induces the expression of multiple stress resistance genes during the intracellular life of Typhimurium.

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2009-09-01
2019-12-13
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Rank product analysis of genes altered in the absence of Toll-like receptor 4 (TLR4) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from TLR4 BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of TLR4 using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of TLR4 using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (50 kb) . Rank product analysis of genes altered in the absence of NADPH-dependent phagosomal oxidase (gp91-phox) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from gp91-phox BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of gp91-phox using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of gp91-phox using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (240 kb) Primers used for construction of . Typhimurium chromosomal deletion mutants [ PDF] (22 kb) Primers used for confirmation of deletion mutants [ PDF] (14 kb) Differential gene expression profiles of . Typhimurium grown in LB and LB+1 mM hydrogen peroxide. Genes shown are those which pass the analysis of variance -test with a Benjamini and Hochberg false discovery rate of 0.05 or less. Genes upregulated more than 3-fold under peroxide stress are coloured red and those downregulated more than 3-fold are shown in blue. [ Excel file] (400 kb)

EXCEL

Rank product analysis of genes altered in the absence of Toll-like receptor 4 (TLR4) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from TLR4 BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of TLR4 using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of TLR4 using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (50 kb) . Rank product analysis of genes altered in the absence of NADPH-dependent phagosomal oxidase (gp91-phox) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from gp91-phox BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of gp91-phox using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of gp91-phox using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (240 kb) Primers used for construction of . Typhimurium chromosomal deletion mutants [ PDF] (22 kb) Primers used for confirmation of deletion mutants [ PDF] (14 kb) Differential gene expression profiles of . Typhimurium grown in LB and LB+1 mM hydrogen peroxide. Genes shown are those which pass the analysis of variance -test with a Benjamini and Hochberg false discovery rate of 0.05 or less. Genes upregulated more than 3-fold under peroxide stress are coloured red and those downregulated more than 3-fold are shown in blue. [ Excel file] (400 kb)

EXCEL

Rank product analysis of genes altered in the absence of Toll-like receptor 4 (TLR4) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from TLR4 BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of TLR4 using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of TLR4 using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (50 kb) . Rank product analysis of genes altered in the absence of NADPH-dependent phagosomal oxidase (gp91-phox) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from gp91-phox BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of gp91-phox using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of gp91-phox using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (240 kb) Primers used for construction of . Typhimurium chromosomal deletion mutants [ PDF] (22 kb) Primers used for confirmation of deletion mutants [ PDF] (14 kb) Differential gene expression profiles of . Typhimurium grown in LB and LB+1 mM hydrogen peroxide. Genes shown are those which pass the analysis of variance -test with a Benjamini and Hochberg false discovery rate of 0.05 or less. Genes upregulated more than 3-fold under peroxide stress are coloured red and those downregulated more than 3-fold are shown in blue. [ Excel file] (400 kb)

PDF

Rank product analysis of genes altered in the absence of Toll-like receptor 4 (TLR4) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from TLR4 BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of TLR4 using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of TLR4 using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (50 kb) . Rank product analysis of genes altered in the absence of NADPH-dependent phagosomal oxidase (gp91-phox) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from gp91-phox BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of gp91-phox using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of gp91-phox using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (240 kb) Primers used for construction of . Typhimurium chromosomal deletion mutants [ PDF] (22 kb) Primers used for confirmation of deletion mutants [ PDF] (14 kb) Differential gene expression profiles of . Typhimurium grown in LB and LB+1 mM hydrogen peroxide. Genes shown are those which pass the analysis of variance -test with a Benjamini and Hochberg false discovery rate of 0.05 or less. Genes upregulated more than 3-fold under peroxide stress are coloured red and those downregulated more than 3-fold are shown in blue. [ Excel file] (400 kb)

PDF

Rank product analysis of genes altered in the absence of Toll-like receptor 4 (TLR4) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from TLR4 BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of TLR4 using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of TLR4 using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (50 kb) . Rank product analysis of genes altered in the absence of NADPH-dependent phagosomal oxidase (gp91-phox) inside primary monocytes (BMDM), shown as fold change in the gene expression levels of bacteria isolated from gp91-phox BMDM compared to those of bacteria isolated from WT BMDM. (A) Genes that are upregulated in absence of gp91-phox using an FDR of 1% or less, with those upregulated 3-fold or more highlighted in red. (B) Genes that are downregulated in absence of gp91-phox using an FDR of 1% or less, with those downregulated 3-fold or more highlighted in blue. [ Excel file] (240 kb) Primers used for construction of . Typhimurium chromosomal deletion mutants [ PDF] (22 kb) Primers used for confirmation of deletion mutants [ PDF] (14 kb) Differential gene expression profiles of . Typhimurium grown in LB and LB+1 mM hydrogen peroxide. Genes shown are those which pass the analysis of variance -test with a Benjamini and Hochberg false discovery rate of 0.05 or less. Genes upregulated more than 3-fold under peroxide stress are coloured red and those downregulated more than 3-fold are shown in blue. [ Excel file] (400 kb)

EXCEL
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