Abstract:BackgroundPurified protein derivative (PPD) has been used for more than half a century as an antigen for the diagnosis of tuberculosis infection based on delayed type hypersensitivity. Although designated as “purified,” in reality, the composition of PPD is highly complex and remains ill-defined. In this report, high resolution mass spectrometry was applied to understand the complexity of its constituent components. A comparative proteomic analysis of various PPD preparations and their functional characterizat… Show more
“…Finally, Additional file 1: Table S1 shows a comparative protein expression profile with two previous bPPD proteomic analysis [18, 19]. Furthermore, our proteomic profile shared 158 proteins with the proteome of M. tuberculosis PPD (MtbPPD) investigated by Prasad et al [17] who were able to identify 265 proteins, the largest number of proteins found in MtbPPD so far.Fig. 1Total number of proteins identified within four bPPD preparationsFig.…”
Section: Resultsmentioning
confidence: 66%
“…More defined knowledge on PPD composition and contribution of individual antigens in TST would give a better insight into the molecular mechanism behind the complex would therefore allow a better selection of proteins specific to M. tuberculosis [17]. Therefore, the identification of the molecular composition would facilitate the development of a more refined reagent [15].…”
BackgroundTuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB).MethodsProteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC–MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system.ResultsThree hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920.ConclusionsThis study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-017-1172-1) contains supplementary material, which is available to authorized users.
“…Finally, Additional file 1: Table S1 shows a comparative protein expression profile with two previous bPPD proteomic analysis [18, 19]. Furthermore, our proteomic profile shared 158 proteins with the proteome of M. tuberculosis PPD (MtbPPD) investigated by Prasad et al [17] who were able to identify 265 proteins, the largest number of proteins found in MtbPPD so far.Fig. 1Total number of proteins identified within four bPPD preparationsFig.…”
Section: Resultsmentioning
confidence: 66%
“…More defined knowledge on PPD composition and contribution of individual antigens in TST would give a better insight into the molecular mechanism behind the complex would therefore allow a better selection of proteins specific to M. tuberculosis [17]. Therefore, the identification of the molecular composition would facilitate the development of a more refined reagent [15].…”
BackgroundTuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB).MethodsProteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC–MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system.ResultsThree hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920.ConclusionsThis study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-017-1172-1) contains supplementary material, which is available to authorized users.
“…Thus, we investigated CD4+ T cell production of two Th1 cytokine mRNAs, IFNG and TNFA, following stimulation of peripheral blood mononuclear cells (PBMC) from donors diagnosed with either active TB or LTBI. As M. tuberculosis antigen we used purified protein derivative (PPD), a complex, multi-epitope protein mixture ( 27 ), to capture a broad T cell response repertoire, which may be more representative of infection-state specific responses than the response to a small number of epitopes ( 28 , 29 ). To capture temporal aspects of the response, antigen stimulation was conducted for 2 and 6 h.…”
Despite advances in diagnosing latent Mycobacterium tuberculosis infection (LTBI), we still lack a diagnostic test that differentiates LTBI from active tuberculosis (TB) or predicts the risk of progression to active disease. One reason for the absence of such a test may be the failure of current assays to capture the dynamic complexities of the immune responses associated with various stages of TB, since these assays measure only a single parameter (release of IFN-γ) and rely on prolonged (overnight) T cell stimulation. We describe a novel, semi-automated RNA flow cytometry assay to determine whether immunological differences can be identified between LTBI and active TB. We analyzed antigen-induced expression of Th1 cytokine mRNA after short (2- and 6-h) stimulation with antigen, in the context of memory T cell immunophenotyping. IFNG and TNFA mRNA induction was detectable in CD4+ T cells after only 2 h of ex vivo stimulation. Moreover, IFNG- and TNFA-expressing CD4+ T cells (Th1 cells) were more frequent in active TB than in LTBI, a difference that is undetectable with conventional, protein-based cytokine assays. We also found that active TB was associated with higher ratios of effector memory to central memory Th1 cells than LTBI. This effector memory phenotype of active TB was associated with increased T cell differentiation, as defined by loss of the CD27 marker, but not with T cell exhaustion, as determined by PD-1 abundance. These results indicate that single-cell-based, mRNA measurements may help identify time-dependent, quantitative differences in T cell functional status between latent infection and active tuberculosis.
“…These results are consistent with previous proteomics work highlighting the importance of the chaperones DnaK, GroEL, and GroES as well as elongation factor Tu and acyl carrier protein as sources of cross-reactivity [ 21 , 22 , 30 ]. Chaperones, which are conserved among most mycobacteria and other bacteria that cause respiratory disease [ 31 ], play important roles in humoral and cellular innate and adaptive immune responses [ 32 , 33 ]. Our results, then, are consistent with work suggesting that false-positive serodiagnosis of tuberculosis tends to reflect immune responses due to homologous vaccination antigens or environmental mycobacteria [ 34 , 35 ].…”
BackgroundBovine purified protein derivative (bPPD) and avian purified protein derivative (aPPD) are widely used for bovine tuberculosis diagnosis. However, little is known about their qualitative and quantitative characteristics, which makes their standardisation difficult. In addition, bPPD can give false-positive tuberculosis results because of sequence homology between Mycobacterium bovis (M. bovis) and M. avium proteins. Thus, the objective of this study was to carry out a proteomic characterisation of bPPD, aPPD and an immunopurified subcomplex from bPPD called P22 in order to identify proteins contributing to cross-reactivity among these three products in tuberculosis diagnosis.MethodsTrypsin digests of bPPD, aPPD and P22 were analysed by nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry. Mice were immunised with bPPD or aPPD, and their serum was tested by indirect ELISA for reactivity against these preparations as well as against P22.ResultsA total of 456 proteins were identified in bPPD, 1019 in aPPD and 118 in P22; 146 of these proteins were shared by bPPD and aPPD, and 43 were present in all three preparations. Candidate proteins that may cause cross-reactivity between bPPD and aPPD were identified based on protein abundance and antigenic propensity. Serum reactivity experiments indicated that P22 may provide greater specificity than bPPD with similar sensitivity for ELISA-type detection of antibodies against M. tuberculosis complex.ConclusionThe subpreparation from bPPD called P22 may be an alternative to bPPD for serodiagnosis of bovine tuberculosis, since it shares fewer proteins with aPPD than bPPD does, reducing risk of cross-reactivity with anti-M. avium antibodies.Electronic supplementary materialThe online version of this article (10.1186/s12014-017-9171-z) contains supplementary material, which is available to authorized users.
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