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Abstract
The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.
Highlights
The current standard for diagnosis of TB in high burden settings is sputum acid-fast bacilli smear microscopy
Preliminary Biomarker Candidate Selection In designing the Multiple Reaction Monitoring (MRM) assays we set out to confirm candidate protein biomarkers identified in three previous studies, including a screening of exosomes isolated from Mtb-infected macrophages, mice and humans
25 candidate proteins were derived from our pilot discovery dataset in which mycobacterial proteins were identified by LCMS/MS in exosomes isolated from Mtb-infected J774a.1 murine macrophage cells (Table 1) [12]; six of which, including the antigen 85 complex (Ag85), GroES, and CFP10, were confirmed by western blot
Summary
The current standard for diagnosis of TB in high burden settings is sputum acid-fast bacilli smear microscopy. While this method identifies individuals with active disease at highest risk of transmitting Mtb, it has a sensitivity as low as 50% [1]. This problem is intensified in patients who are unable to produce sputum such as children and human immunodeficiency virus (HIV) infected patients, two groups who are highly susceptible to TB and who suffer high morbidity and mortality from TB [2,3,4]. Existing serodiagnostics perform so poorly that the World Health
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