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Transcriptional regulation of Chlamydia pneumoniae under hypoxia

ABSTRACT: Purpose: To further define the enhanced metabolic activity of Chlamydia pneumoniae under hypoxia, a transcriptome screen was performed. Next-genration sequencing (NGS) has revolutioned systems-based analysis of transcriptomic pathways. The goals of this study are to compare the transcriptomic profile of C. pneumoniae, grown within HEp-2 cells under normoxic and hypoxic conditions. Methods: Total RNA of C. pneumoniae infected HEp-2 cells cultured under normoxia (20% O2) or hypoxia (2% O2) was isolated by NucleoSpin RNA kit (Macherey Nagel). Human rRNA was depleted by RiboZero rRNA removal kit (Epicentre) in order to enrich bacterial RNA. The tagged cDNA libraries, in the size range of 250 –400 bp, were pooled and single-read sequencing (read length 50 bp) was performed on Illumina HiSeq 2000 by BGI-Hong Kong. Illumina reads were mapped to the Chlamydia genome (GeneBank, version AE001363.1) by TopHat (version TopHat v1.0.12. Gene expression was determined by the Htseq package and data was normalized using the RPKM conversion. Finally, the differential expression analysis was done using the Bioconductor package NOISeq version 2.6.0. The NOISeq-sim function included in the package allows for differential expression estimates in absence of replication by simulating replicates considering that reads counts follow a multinominal distribution. Results: Ranking of differentially expressed chlamydial genes by NOISeq-sim revealed 153 upregulated and 18 downregulated candidate genes. The expression profile of selected genes was validated using qRT-PCR. Most of the upregulated genes under hypoxia belong to the transcriptional and translational machinery or have unknown function. Moreover, transporters show increased expression under hypoxia such as the ATP/ADP translocase (Cpn0614) responsible for NAD uptake. Within the group of metabolic genes, numerous genes belonging to the energy metabolism and nucleotide metabolism were upregulated under hypoxia. Furthermore, thioredoxin reductase (trxB) belonging to the chlamydial redox system was upregulated. Conclusions: Our study represents the first detailed transcriptomic analysis of C. pneumoniae infected HEp-2 cells under hypoxic conditions, generated by RNA-seq technology. Our results show an increased expression of trxB, a part of the chlamydial redox system, which might reduce reactive oxygen species (ROS) under hypoxia. Interestingly, infection of hypoxic cells indeed resulted in a decreased ROS generation compared to hypoxic non-infected cells. We conclude that enhanced growth of C. pneumoniae is the result of hypoxia-induced mitochondrial dysfunction and the associated metabolic switch. Particularly, mitochondrial hyperpolarization is a central mechanism which promotes C. pneumoniae growth under hypoxic conditions. Further, our data imply that mitochondrial dysfunction could also play a major role for other intracellular pathogens, since mitochondrial dysfunction usually occurs under hypoxia and could thereby influence intracellular growth.

ORGANISM(S): Chlamydia pneumoniae

PROVIDER: GSE94697 | GEO | 2017/11/24

SECONDARY ACCESSION(S): PRJNA371835

REPOSITORIES: GEO

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Project description:Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been associated with various chronic diseases such as asthma, coronary heart disease and importantly atherosclerosis. Possibly because the pathogen can exist in a persistent form. TNF-a has been reported to induce chlamydial persitence in epithelial cell lines, however the mechanism of TNF-a-induced persistence has not been reported. Moreover, C. pneumoniae persistently infect human dendritic cells (DCs) and activate DCs to produce cytokines including TNF-a. Induction of chlamydial persistence by other cytokines such as IFN-g is known to be due to indoleamine 2,3-dioxygenase (IDO) activity. The present study therefore, investigated whether C. pneumoniae infection can induce IDO activity in dendritic cells, and whether the restriction of chlamydial growth in the DCs by TNF-a is IDO-dependent. Our data indicate that infection of DCs with C. pneumoniae resulted in the induction of IDO expression. Reporting on our use of anti-TNF-a antibody adalimumab and varying concentrations of TNF-a, we further demonstrate that IDO induction following infection of DCs with C. pneumoniae is TNF-a-dependent. The anti-chlamydial activity induced by TNF-a and the expression of chlamydial 16S rRNA gene, euo, groEL1, ftsk and tal genes was correlated with the induction of IDO. Addition of excess amounts of tryptophan to the DC cultures resulted in abrogation of the TNF-a-mediated chlamydial growth restriction. These findings suggest that infection of DCs by C. pneumoniae induces production of functional IDO, which subsequently causes depletion of tryptophan. This may represent a potential mechanism for DCs to restrict bacterial growth in chlamydial infections. Experiment Overall Design: Human monocyte-derived DCs from five heathy donors were infected with C. pneumoniae at multiplicity of infection of 5 for 72 h. Mock-infected (uninfected) DCs were used as a control for the infection. Total RNA from 72 h chlamydiae-infected or mock-infected DCs was isolated with RNeasy Mini Kit (Qiagen, Hilden, Germany) and DNA contamination was removed with RQ1 DNase (Promega). Total RNA from all donors was pooled and the samples were processed and analyzed at the array facility of Helmholtz center for infection biology, Braunschweig. The integrity of the RNA was verified on the Agilent Bioanalyzer system. We used the Affymetrix Gene Chip Human Genome U133 2.0 Plus. Data were processed using Affymetrix software and signal intensity measured was normalized by the Affymetrix MAS5 algorithm. Data were filtered to exclude genes at background levels (non-expressors, signal intensity at 100).

Project description:The activation of the transcription factor Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small-molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high throughput screen led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations but did not affect expression levels of genes outside of the HIF-1 pathway. Lead structure BAY 87-2243 was found to inhibit HIF-1Î± protein accumulation under hypoxic conditions in NSCLC cell line H460 but had no effect on HIF-1Î± protein accumulation and HIF target gene expression in RCC4 cells lacking VHL activity or in H460 cells after inhibition of HIF prolyl hydroxylase activity. BAY 87-2243 had no effect on HIF-Î±-mRNA levels. Antitumor activity of BAY 87-2243 and suppression of HIF-1 target gene expression in vivo was demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial production of reactive oxygen species (ROS) by blocking complex I activity but has no effect on complex III activity. Lowering of mitochondrial ROS production to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors. We used microarrays to detail the global programme of gene expression that is induced in NSCLC cell line H460 upon hypoxia (16 h incubation at 1 % pO2) and evaluated a dose-dependent effect of our HIF-1-pathway inhibitor BAY 87-2243 on genes tthat are affected by hypoxia. Specificity of BAY 87-2243 for the suppression of HIF-1-mediated gene transcription on a genome-wide scale was evaluated by microarray hybridizations using Affymetrix GeneChip Human Gene 1.0 ST arrays. RNA from normoxic H460 cells and from hypoxic H460 cells incubated with 1, 10 and 100 nM BAY 87-2243 respectively was subjected to array hybridization. Of those 30 genes that were most strongly suppressed by 100 nM BAY 87-2243 in hypoxic H460 cells compared to DMSO-treated hypoxic H460 cells, virtually all of them are induced by prior hypoxia and most of these genes have been described in the literature as HIF-1 target genes

Project description:Published molecular profiling studies in patients with lymphoma suggested the influence of hypoxia inducible factor-1 alpha (HIF1α) targets in prognosis of DLBCL. Yet, the role of hypoxia in hematological malignancies remains unclear. We observed that activation of HIF1α resulted in global translation repression during hypoxic stress in DLBCL. Protein translation efficiency as measured using 35S-labeled methionine incorporation revealed a ≥50% reduction in translation upon activation of HIF1α. Importantly, translation was not completely inhibited and expression of clinically correlated hypoxia targets such as GLUT1, HK2, and CYT-C was found to be refractory to translational repression under hypoxia in DLBCL cells. Notably, hypoxic induction of these genes was not observed in normal primary B-cells. Translational repression was coupled with a decrease in mitochondrial function. Screening of primary DLBCL patient samples revealed that expression of HK2, which encodes for the enzyme hexokinase 2, was significantly correlated with DLBCL phenotype. Genetic knockdown studies demonstrated that HK2 is required for promoting growth of DLBCL under hypoxic stress. Altogether, our findings provide strong support for the direct contribution of HK2 in B-cell lymphoma development and suggest that HK2 is a key metabolic driver of the DLBCL phenotype.ne incorporation revealed a ≥50% reduction in translation upon activation of HIF1α. Importantly, translation was not completely blunted and expression of clinically correlated hypoxia targets such as GLUT1, HK2, and CYT-C was found to be refractory to translational repression under hypoxia in DLBCL cells. Notably, hypoxic induction of these genes was not observed in normal primary B-cells. Translational repression was coupled with decrease in mitochondrial function. Screening of DLBCL patient samples identified that expression of HK2, which encodes for the enzyme hexokinase 2, was significantly correlated with DLBCL phenotype. Genetic knockdown studies show that HK2 is required for promoting growth of DLBCL under hypoxic stress. Altogether, our findings provide more definitive proof of direct contribution of HK2 in development of B-cell lymphoma and suggest that HK2 is a key metabolic driver of DLBCL phenotype.

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Transcriptional regulation of Chlamydia pneumoniae under hypoxia

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