Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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The effect of bacterial capsule on macrophage transcriptional responses to Streptococcus Pneumoniae.

ABSTRACT: Streptococcus Pneumoniae capsule is a major virulence factor. The capsule is known to inhibit complement fixation and phagocytes, but its impact on innate immune responses is not known. To address this question, we compared transcriptional responses by human monocyte derived macrophages in responses to wild type S. pneumoniae with isogenic mutant bacteria lacking their capsule. We conducted these experiments with and without macrophage pretreatment with cytochalasin D to evaluate the impact of phagocytosis, and we also made comparisons to mutant S.pneumoniae lacking bacterial lipoprotein which is known to be necessary for maximal innate immune responses.

ORGANISM(S): Homo sapiens

SUBMITTER: Mahdad Noursadeghi

PROVIDER: E-MTAB-5894 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.

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The effect of bacterial capsule on macrophage transcriptional responses to Streptococcus Pneumoniae.

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