Nicole A. Kruh-Garcia

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

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.

Purified protein derivatives of tuberculin – past, present, and future

The tuberculin skin test, which involves monitoring the immune reaction to an injection of purified protein derivative (PPD), has been the most widely used method for detecting infection with Mycobacterium tuberculosis since its development in 1930s. Until recently, the molecular composition of PPD was unknown. This thwarted the discovery of improved skin testing reagents and drastically hindered efforts to define the mechanism of action. Proteomic evaluation of PPD combined with a detailed analysis in the guinea pig model of tuberculosis led to further definition of the molecular composition of PPD. This communication reviews the history and current status of PPD, in addition to describing candidate next-generation PPD reagents, based on the use of an individual protein or protein cocktails.

Antigen 85 variation across lineages of Mycobacterium tuberculosis-implications for vaccine and biomarker success.

UNLABELLED Mycobacterium tuberculosis secretes several hundred proteins; many of which elicit immune responses. As a result, many of these proteins have been explored for their potential as diagnostic and vaccine candidates. Of these, the Antigen 85 complex proteins, represented by Antigen85 A, B, and C, are the most studied from the mycobacterial secretome. However, vaccine constructs exploiting Antigen 85 as the sole antigen repertoire have not experienced the pre-clinical and clinical trials success originally anticipated. Anecdotal and biochemical evidence suggests that differences in protein abundance may explain this phenomenon. Here, biochemical, molecular, and mass spectrometry approaches were used to quantify Antigen 85 among six M. tuberculosis strains from four phylogenetically distinct clades. Our data demonstrates that the greatest variation in Antigen 85 is ascribed to protein quantities, whereas few transcriptional differences were found. In addition, the ratio of Antigen 85 A, to B, to C is conserved within clades and phylogenetic neighbors. In contrast, no such relationship between individual protein quantities was observed, and in the case of Antigen85 B, this variation even extends within biological replicates of individual isolates. The relevance of Antigen 85 protein quantities and vaccine efficacy remains to be defined. BIOLOGICAL SIGNIFICANCE Absolute quantitation via multiple reaction monitoring mass spectrometry was used to determine the exact molar concentrations of Antigen 85A, B, and C; three key immunodominant proteins present in M. tuberculosis. Further, the concentration of these three proteins was compared among various clades of M. tuberculosis, and demonstrated differences in abundance of two of the three proteins. These proteins have been identified as key antigens in multiple vaccine and diagnostic platforms, thus the potential relevance of their abundance in various M. tuberculosis clades to the successful outcome of these interventions is discussed. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Prospective on Mycobacterium tuberculosis proteomics.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains one of the most prevalent human pathogens in the world. Knowledge regarding the bacillis physiology as well as its mechanisms of virulence, immunogenicity, and pathogenesis has increased greatly in the last three decades. However, the function of about one-quarter of the Mtb coding genome and the precise activity and protein networks of most of the Mtb proteins are still unknown. Protein mass spectrometry and a new interest in research toward the field of functional proteomics have given a new light to the study of this bacillus and will be the focus of this review. We will also discuss new perspectives in the proteomics field, in particular targeted mass spectrometry methods and their potential applications in TB research and discovery.

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

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.

Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications

ABSTRACT Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.

Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic

Biomarkers, derived from either the host (e.g. immunological markers, such as cytokines) or infectious agent (e.g. exported products, such as lipoarabinomannan), are indicative not only of disease but also of disease stage, severity and drug failure. Discovering new biomarkers from easily attainable bodily fluids is essential if we are to control tuberculosis, a diseases that kills 1.5 to 2 million individuals a year. The ideal biomarker could be used in the diagnosis and prognosis of disease from any suspect patient regardless of age, immune status or vaccination history. One potential biomarker source is exosomes; small membranebound vesicles released from cells which can be found circulating in the blood, and can be readily harvested for diagnostic testing. Exosomes shed from Mycobacterium tuberculosis (Mtb) infected samples (from in vitro produced to in vivo models and clinical samples) could provide an ideal reservoir of tuberculosis biomarkers. While it is possible to detect and monitor host and other bacterial components on exosomes, the scope of the following experiments are focused specifically on the Mtb proteins that are incorporated into exosomes.

Fractionation and analysis of mycobacterial proteins.

The extraction and isolation of native bacterial proteins continue to be valuable technical pursuits in order to understand bacterial physiology, screen for virulence determinants, and describe antigens. In this chapter, methods for the manipulation of whole mycobacterial cells are described in detail. Specifically, the concentration of spent culture filtrate media is described in order to permit separation of soluble, secreted proteins; several discrete separation techniques, including precipitation of protein mixtures with ammonium sulfate and separation of proteins by hydrophobic chromatography are also provided. Similarly, the generation of whole cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol enriched proteins is described. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided, as well as a separation technique (isoelectric focusing) that can be applied to separate hydrophobic proteins. Lastly, two commonly used analytical techniques, in-gel digestion of proteins for LC-MS and analysis of intact proteins by MALDI-ToF MS, are provided for rapid analysis of discrete proteins within subcellular or chromatographic fractions. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins. In addition, a number of these methods may be applied to extract and analyze mycobacterial proteins from cell lines and host derived samples.

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.