Dataset Information

Changes in the membrane proteome of exosomes released from human macrophages after infection with Mycobacterium tuberculosis.

ABSTRACT: One of the greatest obstacles to eliminating TB is the lack of predictive and diagnostic biomarkers. Exosomes are a promising alternative source of biomarkers for TB since they can carry mycobacterial antigens. We hypothesize that exosomes from Mtb-infected macrophages exhibit a characteristic selection of human protein that can be evaluated as TB biomarkers. We looked for the localization—exosomal membrane or lumen—of the differentially abundant proteins. Exosomes were obtained from THP-1-derived macrophages (Mtb infected and uninfected controls). The exosome population was validated by nanoparticle tracking analysis and western blot. The protein composition of exosomes was evaluated by tandem mass spectrometry. Differences in protein composition and abundances between exosomes from infected and control cells were evaluated by t-test the proteomics findings were confirmed by western blot. Using a biotinylation strategy we verified the protein localization. Forty-seven proteins were significantly more abundant in exosomes from Mtb-infected cells, 66% were predicted to be membrane associated. The biotinylation pattern confirmed the membrane-association of some of these proteins.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo sapiens

TISSUE(S): Cell Culture

DISEASE(S): Not Available

SUBMITTER: Gustavo Diaz

PROVIDER: PXD004062 | Pride | 2016-12-20

REPOSITORIES: Pride

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Publications

Changes in the Membrane-Associated Proteins of Exosomes Released from Human Macrophages after Mycobacterium tuberculosis Infection.

Diaz Gustavo G Wolfe Lisa M LM Kruh-Garcia Nicole A NA Dobos Karen M KM

Scientific reports 20161129

Tuberculosis (TB) is the deadliest infectious disease worldwide. One obstacle hindering the elimination of TB is our lack of understanding of host-pathogen interactions. Exosomes, naturally loaded with microbial molecules, are circulating markers of TB. Changes in the host protein composition of exosomes from Mycobacterium tuberculosis (Mtb)-infected cells have not been described, can contribute to our understanding of the disease process, and serve as a direct source of biomarkers or as capture ...[more]

PMID: 27897233

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Project description:Tuberculosis is a deadly infectious disease, which kills millions of people every year. The causative pathogen, Mycobacterium tuberculosis (MTB), is estimated to have infected up to a third of the world’s population; however, only approximately 10% of healthy individuals progress to active TB disease. Despite evidence for heritability, it is not currently possible to predict whether a healthy person is susceptible to TB. To explore approaches to classify susceptibility to TB, we infected with MTB dendritic cells (DCs) from putatively resistant individuals diagnosed with latent TB, and from susceptible individuals that had recovered from a past episode of active TB. We measured genome-wide gene expression levels in infected and non-infected cells and found hundreds of differentially expressed genes between susceptible and resistant individuals in the non-infected cells. We further found that genetic polymorphisms in proximity to the differentially expressed genes between susceptible and resistant individuals are more likely to be associated with TB susceptibility in published GWAS data. By intersecting the gene expression and GWAS data, we identified two promising candidate genes: CCL1 and UNC13A. Lastly, we trained a classifier based on the gene expression levels in the non-infected cells, and demonstrated decent performance on our data and an independent data set. Overall, our promising results from this small study suggest that training a classifier on a larger cohort may enable us to accurately predict TB susceptibility. Overall design: We obtained whole blood samples from 25 healthy male Caucasian individuals. Six of the donors had recovered from active TB, and are thus putatively susceptible. The remaining 19 tested positive for latent TB without ever experiencing symptoms of active TB, and are thus putatively resistant. We isolated dendritic cells (DCs) and treated them with Mycobacterium tuberculosis (MTB) or a mock control for 18 hours. To measure genome-wide gene expression levels in infected and non-infected samples, we isolated and sequenced RNA using a processing pipeline designed to minimize the introduction of unwanted technical variation. There are a total of 50 samples (25 individuals x 2 treatments).