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RNA-seq profiling of parental and NLUCAT1-deleted A549 cells under hypoxia

ABSTRACT: RNA-seq profiling of parental and NLUCAT1-deleted A549 cells under hypoxia

PROVIDER: PRJNA480897 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7516678_1.fastq.gz	Fastqsanger.gz
	SRR7516678_2.fastq.gz	Fastqsanger.gz
	SRR7516679_1.fastq.gz	Fastqsanger.gz
	SRR7516679_2.fastq.gz	Fastqsanger.gz

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Project description: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.

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RNA-seq profiling of parental and NLUCAT1-deleted A549 cells under hypoxia

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