BackgroundThe spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection.Methodology/Principal FindingsTo better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance.ConclusionsThis is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.

Neuroglobin is a heme protein of which increased levels provide neuroprotection against amyloid proteinopathy and hemorrhagic damage. These cellular stressors involve the promotion of ferroptosis—an iron-dependent, lipid peroxide-accreting form of cell death. Hence, we questioned whether neuroglobin could oppose ferroptosis initiation. We detected human neuroglobin (hNgb)-EGFP-expressing SH-SY5Y cells to be significantly more resistant to ferroptosis induction, identifying 0.68-fold less cell death. To elucidate the underlying pathways, this study investigated hNgb-protein interactions with a Co-IP-MS/MS approach both under a physiological and a ferroptotic condition. hNgb binds to proteins of the cellular iron metabolism (e.g., RPL15 and PCBP3) in an unstressed condition and shows an elevated binding ratio towards cell death-linked proteins, such as HNRNPA3, FAM120A, and ABRAXAS2, under ferroptotic stress. Our data also reveal a constitutive interaction between hNgb and the longevity-associated heterodimer XRCC5/XRCC6. Disentangling the involvement of hNgb and its binding partners in cellular processes, using Ingenuity Pathway Analysis, resulted in the integration of hNgb in the ubiquitination pathway, mTOR signaling, 14-3-3-mediated signaling, and the glycolysis cascade. We also detected a previously unknown strong link with motor neuropathies. Hence, this study contributes to the elucidation of neuroglobin’s involvement in cellular mechanisms that provide neuroprotection and the upkeep of homeostasis.

A diagnostic test that could detect Treponema pallidum antigens in urine would facilitate the prompt diagnosis of syphilis. Materials & methods: Urine from 54 individuals with various clinical stages of syphilis and 6 controls were pooled according to disease stage and interrogated with complementary mass spectrometry techniques to uncover potential syphilis biomarkers. Results & conclusion: In total, 26 unique peptides were uncovered corresponding to four unique T. pallidum proteins that have low genetic sequence similarity to other prokaryotes and human proteins. This is the first account of direct T. pallidum protein detection in human clinical samples using mass spectrometry. The implications of these findings for future diagnostic test development is discussed. Data are available via ProteomeXchange with identifier PXD009707.