Candidate Treponema pallidum biomarkers uncovered in human urine samples from MSM individuals with syphilis using a multi-platform mass spectrometry approach

ABSTRACT: Background A molecular diagnostic test that could directly evaluate Treponema pallidum subspecies pallidum antigens in urine would greatly facilitate the prompt diagnosis of syphilis. Methods A multi-platform high resolution shotgun proteomics approach was used to investigate the presence of T. pallidum proteins in male urine samples collected from individuals with and without syphilis. Study participants were included in an observational cohort study conducted at the Institute of Tropical Medicine, Antwerp, Belgium from 2014 to 2017. In total, N = 60 individuals were included in this sub-study, including n = 36 with early symptomatic stage (primary/secondary), n = 13 early latent and n = 5 late latent stage and n = 6 controls. Almost all participants were HIV-positive (56/60; 93 %) and identified as men who have sex with men (MSM) (59/60; 98 %). Sixteen (27 %) individuals were serum PCR positive for T. pallidum at the time of diagnosis. Ten pools containing urine from six participants (±1.5 mL per individual) were created based on clinical staging. Urine protein was extracted from the pools by a two-step molecular weight cut-off centrifugal filtration to remove large proteins and non-protein material. Samples were digested, subjected to multi-dimensional liquid chromatographic separation based on hydrophobicity and were analysed with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and/or liquid chromatography- electrospray ionization quadruple ion mobility time-of-flight high-definition mass spectrometry (LC/ESI-Q-IM-TOF) in data dependent (MALDI-TOF/TOF) and data independent HDMSE (ESI-IM-Q-TOF) acquisition modes. Results In total, 26 peptides corresponding to four unique T. pallidum proteins were uncovered in four different pooled urine samples, from n = 3 symptomatic (primary/secondary stage; PSS) and n = 1 latent stage pool. Peptides from GTPase Obg (Tp0742) were detected in two separate PSS pooled samples. Protein designations are listed as in the currently annotated T. pallidum proteome available at the UniProt database (Proteome ID: UP000014259) which is related to the genome assembly and annotation ID GCA_000410535.2 listed on the European Nucleotide Archive. Eleven peptides from an uncharacterized protein (Tp0369) and 8 peptides from a protein annotated as the Borrelia-like p83/100 protein (Tp0486) were detected in two different PSS pooled samples. A protein annotated as an ABC superfamily transport protein (Tp0804) was uncovered in a latent stage pooled sample. BLAST analyses revealed low peptide sequence similarity with other pathogenic and commensal prokaryotes and human proteins. Conclusions High resolution mass spectrometric analyses of protein extracts from pooled urine samples resulted in the detection of four T. pallidum proteins. This is the first successful account of applying proteomics approaches to detect treponemal antigens in clinical samples. These proteins could represent potential biomarkers for downstream diagnostic applications.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

DISEASE(S): Syphilis

SUBMITTER: Maarten Dhaenens  

LAB HEAD: Dieter Deforce

PROVIDER: PXD009707 | Pride | 2018-10-18

REPOSITORIES: Pride