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Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis.

ABSTRACT: Background: Current syphilis diagnostic strategies are lacking a sensitive manner of directly detecting Treponema pallidum antigens. A diagnostic test that could directly detect T. pallidum antigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up. Methods: In this study, 11 candidate T. pallidum biomarker proteins were chosen according to their physiochemical characteristics, T. pallidum specificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer. Results: No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purified T. pallidum were prepared in PBS. Polyclonal anti- T. pallidum antibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300 T. pallidum/ml in PBS. Conclusions: Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration of T. pallidum proteins. Alternative sample preparation strategies may improve the detectability of T. pallidum proteins in biofluids.

SUBMITTER: Van Raemdonck GA

PROVIDER: S-EPMC6248270 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis.

Van Raemdonck Geert A GA Osbak Kara K KK Van Ostade Xaveer X Kenyon Chris R CR

F1000Research 20180319

Background: Current syphilis diagnostic strategies are lacking a sensitive manner of directly detecting Treponema pallidum antigens. A diagnostic test that could directly detect T. pallidum antigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up. Methods: In this study, 11 candidate T. pallidum biomarker proteins were chosen according to their phys ...[more]

PMID: 30519456

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Project description:Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Project description: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.

Project description: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.

Dataset Information

Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis.

Publications

Needle lost in the haystack: multiple reaction monitoring fails to detect <i>Treponema pallidum</i> candidate protein biomarkers in plasma and urine samples from individuals with syphilis.

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