Proteomic analysis of protein purified derivative of Mycobacterium bovis

Abstract

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.

Proteomic Analysis of Antigen 60 Complex of M. bovis Bacillus Calmette-Guérin Reveals Presence of Extracellular Vesicle Proteins and Predicted Functional Interactions

“…Commercial A60 (S1 and S2) were subjected to shotgun proteomic analysis on LC-MS/MS and searched against BCG Uniprot database. Similar to previous studies of “method-antigen complexes” such as MTB and M. bovis PPD [38,39], shotgun proteomic analysis of the A60-BCG complex antigen preparation detected a high number of proteins, specifically 630 and 595 proteins with ≥2 significant matches in A60 S1 and A60 S2, respectively, of which 440 were common to both samples (71% of A60 S1; 74% of A60 S2), amounting to 778 combined protein matches (Figure 1A) (Table S1). Overall, the proteins identified in the A60 represents approximately 19.9% of the 3891 proteins identified in the BCG proteome [40].…”

“…However, in recent years, there have been emerging interest in re-evaluation of classical mycobacterial antigen preparations using global approaches such as mass spectrometry, which may provide more insights into their characteristics to inform research in biomarkers and vaccine development. These studies have primarily focussed on the proteomic profiling of PPD [38,39], a cocktail of antigens used for determining latent tuberculosis in the Tuberculin Skin/Mantoux Test (including M. bovis PPD, used for diagnosis of bovine TB), which has been historically purified from steaming cultures of MTB by repeated precipitation with ammonium sulfate [41]. Given the crude nature of the PPD and A60, it is unsurprising that the two preparations reportedly share several similar antigens [25].…”

Genomic Polymorphism Associated with the Emergence of Virulent Isolates of Mycobacterium bovis in the Nile Delta

“…2). Overall, all MBE field isolates clustered with M. bovis AN5 originally a Brazilian strain used to produce PPD worldwide 25,26 while the BCG-Like isolates clustered with Russian BCG strain which included the deletion of the RD1 region 19 (Supplemental Fig. 1).…”

Spectroscopic Characterization of Bovine, Avian and Johnin Purified Protein Derivative (PPD) with High-Throughput Fourier Transform InfraRed-Based Method

“…The proteome analysis that was carried out by Santema et al [34], while using LC-MS/MS, showed that PPDs from the Netherlands and Spain share a high range of proteins with a high degree of homology, mainly between closely-related species as MAP and MA. Moreover, as reported by Roperto et al [64], proteome analysis of the PPDB using bottom-up proteomics (protein digestion and nano-LC-MS/MS analysis) showed 198 proteins, which had not already been reported, and the proteomic pattern overlapping 80 proteins with the previous proteomes of the UK and Brazil PPDBs and 139 protein constituents of the Korean PPDBs. When PPDJ, PPDA, and PPDB protein component is compared, specific regions can be identified in these not degraded proteins, which provide pathogen specific immune responses against specific etiologic agents, such as MAP, MA, and MB.…”