Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Whole blood RNA-sequencing from volunteers in a human influenza challenge study, in which blood aliquots were infected and incubated with BCG before and after influenza infection

ABSTRACT: We aimed to test the hypothesis that influenza infection of healthy adults alters whole blood mycobacterial control through modulation of anti-mycobacterial immune responses. We employed a whole blood mycobacterial growth inhibition assay within the framework of a human influenza challenge study. We utilised a recombinant reporter strain of mycobacteria, Mycobacterium bovis Bacille Calmette Guerin (BCG) lux, whose luminescence provides a quantitative read-out of live mycobacteria present. Volunteers were challenged with Influenza A (H3N2) virus and infection confirmed by qPCR of nasal lavage samples. Blood aliquots, taken from participants before and after influenza infection, were infected with BCG lux and incubated for up to 72 hours (0, 6, 24, 72 h). BCG-uninfected blood aliquots were also incubated. Whole blood mycobacterial growth and transcriptomic, cytokine and cellular responses to mycobacterial infection were measured in parallel and compared in the same subjects, before versus after influenza infection.

INSTRUMENT(S): R, Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Claire Broderick

PROVIDER: E-MTAB-15137 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Pathogenic mycobacteria, such as Mycobacterium tuberculosis, modulate the host immune system to evade clearance and promote long-term persistence, resulting in disease progression or latent infection. Understanding the mechanisms pathogenic mycobacteria use to evade the host immune system is critical to better understanding the pathogenesis of mycobacterial infection. Protein kinase G (PknG) in pathogenic mycobacteria has been shown to play an important role in avoiding clearance by macrophages through blocking phagosome-lysosome fusion; however, the exact mechanism is not completely understood. Here, to investigate the influence of mycobacterial PknG on the phosphoprotein signalling cascades triggered during early events prior to phagocytosis, RAW 264.7 macrophage cell lines were stimulated with M. bovis BCG wild-type and PknG knock-out mutant strains and harvested prior to uptake. After proteolysis, phosphopeptides were enriched via TiO2 columns and subjected to LC-MS/MS to identify differentially phosphorylated peptides between macrophages exposed to the wild-type and PknG knock-out mutant. A total of 1401 phosphosites on 914 unique host proteins were identified. Following phosphoproteome normalisation and differential expression analysis, a total of 149 phosphosites were differentially phosphorylated in the RAW 264.7 macrophages exposed to the wild-type versus the PknG knock-out mutant. A subset of 95 phosphosites was differentially up-regulated in the presence of PknG. In addition, a presence/absence analysis identified 34 phosphosites on 27 host proteins to be exclusively phosphorylated in the presence of PknG. Functional analysis of our data revealed that PknG kinase activity in mycobacteria primes an altered phosphorylation state in host macrophages prior to establishment of infection, through interfering with host spliceosomal and translational machinery, actin cytoskeletal organisation, pro-inflammatory cytokine induction, and programmed cell death, thus establishing a new role for PknG in directing the fate of mycobacteria in macrophage infections.

Dataset Information

Whole blood RNA-sequencing from volunteers in a human influenza challenge study, in which blood aliquots were infected and incubated with BCG before and after influenza infection

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