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Using circulating microbial cell-free DNA to identify persistent Treponema pallidum infection in serofast syphilis patients.

ABSTRACT: The question of whether serofast status of syphilis patients indicates an ongoing low-grade Treponema pallidum (T. pallidum) infection remains unanswered. To address this, we developed a machine learning model to identify T. pallidum in cell-free DNA (cfDNA) using next-generation sequencing (NGS). Our findings showed that a TP_rate cut-off of 0.033 demonstrated superior diagnostic performance for syphilis, with a specificity of 92.3% and a sensitivity of 71.4% (AUROC = 0.92). This diagnosis model predicted that 20 out of 92 serofast patients had a persistent low-level infection. Based on these predictions, re-treatment was administered to these patients and its efficacy was evaluated. The results showed a statistically significant decrease in RPR titers in the prediction-positive group compared to the prediction-negative group after re-treatment (p < 0.05). These findings provide evidence for the existence of T. pallidum under serofast status and support the use of intensive treatment for serofast patients at higher risk in clinical practice.

SUBMITTER: Wu MY

PROVIDER: S-EPMC10959656 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Using circulating microbial cell-free DNA to identify persistent Treponema pallidum infection in serofast syphilis patients.

Wu Meng Yin MY Chen Lu L Liu Li Cheng LC Liu Ming Juan MJ Li Yan Feng YF Zheng He Yi HY Leng Ling L Zou Yi Jun YJ Chen Wei Jun WJ Li Jun J

iScience 20240301 4

The question of whether serofast status of syphilis patients indicates an ongoing low-grade Treponema pallidum (T. pallidum) infection remains unanswered. To address this, we developed a machine learning model to identify T. pallidum in cell-free DNA (cfDNA) using next-generation sequencing (NGS). Our findings showed that a TP_rate cut-off of 0.033 demonstrated superior diagnostic performance for syphilis, with a specificity of 92.3% and a sensitivity of 71.4% (AUROC = 0.92). This ...[more]

PMID: 38523794

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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.

Dataset Information

Using circulating microbial cell-free DNA to identify persistent Treponema pallidum infection in serofast syphilis patients.

Publications

Using circulating microbial cell-free DNA to identify persistent <i>Treponema pallidum</i> infection in serofast syphilis patients.

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