Unknown,Transcriptomics,Genomics,Proteomics

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Macrophages recognize the Helicobacter pylori type IV secretion system in the absence of TLR signaling

ABSTRACT: Helicobacter pylori conquered the world by colonizing the human stomach. There are two major groups of strains, one with and one without a cag pathogenicity island (cagPAI), which result in different clinical outcomes with increased severity in cagPAI+ H. pylori infections such as higher inflammation and higher rates of gastric carcinogenesis. Since the gastro-intestinal tract is bombarded with nonspecific ligands that would induce innate signaling, tissue-resident immune cells are believed to lack toll-like receptor (TLR) signaling (anergy).To illuminate how anergic innate immune cells respond to H. pylori, we first investigated the transcriptome of H. pylori infected wild type and MyD88/Trif -/- macrophages, which lack TLR signaling. We observed that the majority of regulated genes in wt macrophages were dependent on TLR signaling. In addition, we found that some of the upregulated genes changed their kinetic behavior depending on TLR signaling. Only anergic macrophages were able to differentiate between cagPAI+ and cagPAI- H. pylori via their transcriptional kinetics of specific early response genes. These genes showed a strong bias towards adenylate-uridylate-rich elements (AREs) containing genes, including the prominent pro-inflammatory cytokines IL-1beta and TNF-alpha. Differentiation was dependent on the presence of the cag type 4 secretion system (cagT4SS), but not the CagA effector protein. Thus, we speculate that the direct recognition of the secretion system by tissue-resident macrophages enables the host to differentiate between pathogenic (cagPAI+) and commensal (cagPAI-) H. pylori variants. Microarray experiments were performed as dual-color hybridizations on Agilent mouse whole genome catalog 44K arrays. To compensate for dye-specific effects, a dye-reversal color-swap was applied. Cells were analyzed at 1h and 3h post infection

ORGANISM(S): Mus musculus

SUBMITTER: Hans-Joachim Mollenkopf

PROVIDER: E-GEOD-56791 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Macrophages recognize the Helicobacter pylori type IV secretion system in the absence of toll-like receptor signalling.

Koch Manuel M Mollenkopf Hans-Joachim HJ Meyer Thomas F TF

Cellular microbiology 20150903 1

Helicobacter pylori strains carrying the cag pathogenicity island (cagPAI) provoke an increased inflammatory response, conferring an increased risk of ulcer formation and carcinogenesis. How the immune system recognizes the presence of cagPAI positive strains is yet unclear. By comparing the transcriptional response of wild type and MyD88/Trif(-/-) bone marrow macrophages to infection with H. pylori, we found that the majority of regulated genes were dependent on toll-like receptor (TLR) signall ...[more]

PMID: 26243717

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Project description:The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Although some Francisella determinants of intracellular growth have been identified, much remains to be understood about the pathogenesis of this organism. In particular, how Francisella responds to its intracellular environment could provide clues about its intracellular biology and reveal pathogenic determinants based on their intracellular expression profiles. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages. Phagocytosed bacteria rapidly responded to their intracellular environment and progressively altered their transcriptional profile over time. Differential gene expression profiles were revealed that correlated with specific intracellular locations of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of a subset of transport and metabolic genes characterized the cytosolic replication stages. Expression of the Francisella Pathogenicity Island (FPI) genes, the functions of which are associated with intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci putatively encoding hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated during the intracellular cycle. In-frame deletion of FTT0383, the Schu S4 ortholog of fevR, of FTT0369c or FTT1676 abolished the ability of Schu S4 to either survive or proliferate intracellularly, demonstrating that bacterial factors of intracellular pathogenesis can be identified based on their intracellular expression profile. In conclusion, establishing the intracellular transcriptome of Francisella has revealed important aspects of its intracellular biology and identified novel virulence determinants of this pathogen. Keywords: Time series Intracellular cycle within primary murine macrophages using the time series of zero, one, two, four, eight, twelve, sixteen and twenty-four hours

Project description:The transposon site hybridization (TraSH) technique (Sassetti, CM et al. 2001. PNAS 98:12712-7) was utilized to identify genes important for the survival of Y. pestis within murine macrophages. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool) in order to identify Y. pestis genes important for survival in macrophages. Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs can be used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool). Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs was used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe.

Dataset Information

Macrophages recognize the Helicobacter pylori type IV secretion system in the absence of TLR signaling

Publications

Macrophages recognize the Helicobacter pylori type IV secretion system in the absence of toll-like receptor signalling.

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