Deep proteome coverage advances knowledge of Treponema pallidum protein expression profiles during infection

Abstract

Comprehensive proteome-wide analysis of the syphilis spirochete, Treponema pallidum ssp. pallidum, is technically challenging due to high sample complexity, difficulties with obtaining sufficient quantities of bacteria for analysis, and the inherent fragility of the T. pallidum cell envelope which further complicates proteomic identification of rare T. pallidum outer membrane proteins (OMPs). The main aim of the present study was to gain a deeper understanding of the T. pallidum global proteome expression profile under infection conditions. This will corroborate and extend genome annotations, identify protein modifications that are unable to be predicted at the genomic or transcriptomic levels, and provide a foundational knowledge of the T. pallidum protein expression repertoire. Here we describe the optimization of a T. pallidum-specific sample preparation workflow and mass spectrometry-based proteomics pipeline which allowed for the detection of 77% of the T. pallidum protein repertoire under infection conditions. When combined with prior studies, this brings the overall coverage of the T. pallidum proteome to almost 90%. These investigations identified 27 known/predicted OMPs, including potential vaccine candidates, and detected expression of 11 potential OMPs under infection conditions for the first time. The optimized pipeline provides a robust and reproducible workflow for investigating T. pallidum protein expression during infection. Importantly, the combined results provide the deepest coverage of the T. pallidum proteome to date.

In-Depth Proteome Coverage of In Vitro-Cultured Treponema pallidum and Quantitative Comparison Analyses with In Vivo-Grown Treponemes

“…The correlation between in vivo and in vitro PPRP expression is further highlighted by the finding that only TPANIC_0225, TPANIC_0789, and TPANIC_0862 were identified as highabundance proteins from in vivo-grown T. pallidum. 25 These findings further suggest that a low expression level of treponemal proteins that are important for T. pallidum pathogenesis (PPRPs) and/or exposed to the host (OMPs) is a host detection avoidance strategy that may contribute to the welldocumented persistent nature of T. pallidum. 10,11 Overall, our comparative analyses show that the protein expression profiles of treponemes from both in vivo and in vitro cultures are similar.…”

“…All MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, USA; version IseNode in Proteome Discoverer 2.5.0.400) containing a single customized database consisting of 1261 T. pallidum proteins (Nichols strain, NCBI reference sequence NC_021490, all proteome annotation revisions from June 17, 2013 to July 4, 2021), 895 rabbit proteins (UniProt Oryctolagus cuniculus proteome, UP000001811), and common contaminants [https://www.thegpm.org/crap/] (Table S1). Similar to our previous in vivo-grown T. pallidum global proteomics study, 25 searches for T. pallidum and rabbit proteins were performed simultaneously using the combined T. pallidum/rabbit protein database. Database search parameters were the same as those of previous in vivo-grown T. pallidum studies: 25,27 precursor tolerance (parent ion tolerance) = 10 ppm; MS/MS tolerance (fragment ion mass tolerance) = 0.6 Da; enzyme specificity = trypsin, with a maximum of two missed cleavages allowed; instrument type = ESI-TRAP; fixed modification = carbamidomethylation (C); and variable modifications = acetylation of the peptide N-terminus and oxidation (M).…”

“…LPS-assembly protein LptD present, Houston 25 16, 68, and 71 TPANIC_0548 UPF0164 family protein present, Houston 25 16, 52, 68, and 71 TPANIC_0557 DUF1007 family protein present, Houston 25…”

“…major outer sheath N-terminal domain-containing protein, TprI present, Houston, 25 MSP porin, TprK present, Houston 25 52 and 63 TPANIC_0923…”

“…Global transcriptomics studies performed on T. pallidum, using DNA microarray and RNA-seq methodologies, have shown that all predicted protein-coding genes are expressed at the transcript level. , Parallel global proteomics studies have resulted in less protein expression coverage, − having been hindered by the historical requirement for growth of T. pallidum in rabbit testes (in vivo-grown T.…”