Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS

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.

Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages

“…Taken together, our results indicate the BMVs, not host-derived exosomes, are the EV population responsible for the trafficking of Mtb molecules from infected macrophages in vitro and for the regulation of uninfected immune cells. Interestingly, EVs (believed to be exosomes) that contain Mtb antigens have been isolated from the serum of patients with latent and active TB; several of these antigens have also been identified in Mtb BMVs (23, 35). This demonstrates that the release of EVs bearing Mtb components from Mtb-infected cells is physiologically relevant and occurs in the course of human disease.…”

Mycobacterium tuberculosis Membrane Vesicles Inhibit T Cell Activation

“…M. tuberculosis produces BVs both during macrophage infection and in axenic culture; the BVs produced under these two conditions carry overlapping content (1–3, 10–12) and similar immune-modulatory properties (3, 12–14). The content and immune-modulatory properties of exosome preparations from infected macrophages (1, 5, 10) are also overlapping with BVs (11, 12, 15), although our interpretation is that this is due to the presence of BVs in the exosome preparations (3).…”

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

“…In the first scenario, several studies have demonstrated that exosomes released from Mtb -infected cells contain mycobacterial products including proteins, RNAs and glycolipid141516. More importantly, our laboratory recently published the discovery of a panel of Mtb peptides that were identified in exosomes isolated from the sera of TB patients and LTBI individuals, confirming the fact that Mtb infection modifies exosome composition and could be a source of biomarkers for TB17.…”

“…Exosomes are an important source of biomarkers for TB by virtue of the discovery of mycobacterial molecules packaged within these vesicles14151727. Here, the effect of the infection on host exosomal protein composition illustrates another potential opportunity to exploit exosomes as biomarkers of TB.…”