Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of cytoplasmic and nucleoplasmic samples of a time-course of Ad5-infected A549 cells

ABSTRACT: Interested in how Adenovirus infection regulates temporal changes and differential export of viral and cellular mRNAs, we infected A549 cells with HAdV-C5 (H5pg4100) at 6, 12, 24 and 48 hours post-infection. Cells were infected with a MOI of 30 ffu and a non-infected (mock) sample was also included. After the infection period, cells were collected by trypsinization and fractionated to separate nucleus and cytoplasm with an NP-40 lysis buffer and RNA was extracted from each sample with TRIzol, followed by a column extraction using the RNeasy Plus Mini Kit. The cDNA libraries were made from mRNAs by first performing a poly(A) selection to the total RNA sample. The cDNA libraries were generated with the ScriptSeq v2 RNAseq Kit using their strand-specific protocol and a multiplex-sequencing was performed on the HiSeq 2500 platform using a paired-end run with a depth of approximately 50 million reads per sample. Two biological replicates of each infection time point and cellular fraction were sequenced using three technical replicates.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Margarita Valdes Aleman

PROVIDER: E-MTAB-12386 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Objectives: Therapies being safe, effective and not vulnerable to develop resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by the use of host-directed drugs to counteract microbial infections. This study sought to identify cellular genes and pathways differentially expressed during airways epithelial infection by nontypable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). Based on the proposed relationship between NTHi intracellular location and persistence, the antimicrobial potential of a panel of drugs perturbing host pathways used by NTHi to enter epithelial cells was investigated. Methods: We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing A549 cell whole genome expression profiling, and identified a panel of host target candidates which were pharmacologically modulated. Interfering drugs were tested for their bactericidal effect, cytotoxicity, effect on the interplay NTHi-epithelial cell, and effect on NTHi respiratory infection in vivo, by assessing lung bacterial loads in a murine intranasal infection model. Results: The sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi; the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts both intracellularly and in the lungs of infected mice. Conclusions: PDE4 inhibition is currently prescribed in COPD; resveratrol is a geroprotector attractive for COPD treatment by preventing lung aging. This work provides evidence for the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection. Haemophilus influenzae clinical strain NTHi375 induced gene expression in A549 human type II pneumocyte cell line was measured at 2 h post-infection. Non infected cells were used as a control. Four independent experiments were performed for each condition.

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RNA-seq of cytoplasmic and nucleoplasmic samples of a time-course of Ad5-infected A549 cells

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