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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.

Tuberculosis (TB) remains a public health issue causing millions of infections every year. Of these, about 15% ultimately result in death. Efforts to control TB include development of new and more effective vaccines, novel and more effective drug treatments, and new diagnostics that test for both latent TB Infection and TB disease. All of these areas of research benefit from a good understanding of the physiology of Mycobacterium tuberculosis (Mtb), the primary causative agent of TB. Mtb secreted protein antigens have been the focus of vaccine and diagnosis research for the past century. Recently, the discovery of extracellular vesicles (EVs) as an important source of secreted antigens in Mtb has gained attention. Similarly, the discovery that host EVs can carry Mtb products during in vitro and in vivo infection has spiked interest because of its potential use in blood-based diagnostics. Despite advances in understanding the content of Mtb and Mtb-infected host extracellular vesicles, our understanding on the biogenesis and role of Mtb and host extracellular vesicles during Mtb infection is still nascent. Here, we explore the current literature on extracellular vesicles regarding Mtb, discuss the host and Mtb extracellular vesicles as distinct entities, and discuss current gaps in the field.

Background Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins. Methods Multiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m / z interference. The final optimized method was tested in 40 exosome samples from TB positive patients. Results Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region. Conclusions An enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.

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 targets to enrich for exosomes containing microbial molecules. Here, the protein composition of exosomes from Mtb -infected and uninfected THP-1-derived macrophages was evaluated by tandem-mass-spectrometry and differences in protein abundances were assessed. Our results show that infection with Mtb leads to significant changes in the protein composition of exosomes. Specifically, 41 proteins were significantly more abundant in exosomes from Mtb- infected cells; 63% of these were predicted to be membrane associated. Thus, we used a novel biotinylation strategy to verify protein localization, and confirmed the localization of some of these proteins in the exosomal membrane. Our findings reveal another important scenario where Mtb could be influencing changes in host cells that unveil new features of the host-pathogen interaction and may also be exploited as a source of biomarkers for TB.

Abstract Circulating extracellular vesicles (EVs) are biomarkers of and contributors to the etiology of disease. Skeletal muscle (SkM) and white adipose tissue (WAT) are the two largest organs by mass in humans and rodents but the relative contribution of EVs from these tissues is unknown. We hypothesized that SkM tissue secretes more EVs than WAT and that a dual fluorescent reporter mouse could be used to detect SkM myofiber-derived EVs in vivo. Human Protein Atlas data and directly measuring EV secretion in mouse SkM and WAT using an ex vivo tissue explant model confirmed that SkM tissue secretes more EVs than WAT. Differences in EV secretion between SkM and WAT were not due to SkM contraction but may be explained by differences in tissue metabolic capacity. A SkM myofiber-specific dual fluorescent reporter mouse was created. Spectral flow cytometry revealed that SkM myofibers are a major source of SkM tissue-derived EVs ex vivo but few reach the circulation in vivo. Our findings demonstrate that SkM secretes more EVs than WAT and many come from SkM myofibers, but our in vivo data indicate that EVs secreted by SkM myofibers may remain primarily in their local extracellular environment. Competing Interest Statement The authors have declared no competing interest.