Dataset Information

Resequencing of Treponema pallidum ssp. pallidum strains Nichols and SS14: correction of sequencing errors resulted in increased separation of syphilis treponeme subclusters.

ABSTRACT:

Background

Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, is a highly clonal bacterium showing minimal genetic variability in the genome sequence of individual strains. Nevertheless, genetically characterized syphilis strains can be clearly divided into two groups, Nichols-like strains and SS14-like strains. TPA Nichols and SS14 strains were completely sequenced in 1998 and 2008, respectively. Since publication of their complete genome sequences, a number of sequencing errors in each genome have been reported. Therefore, we have resequenced TPA Nichols and SS14 strains using next-generation sequencing techniques.

Methodology/principal findings

The genomes of TPA strains Nichols and SS14 were resequenced using the 454 and Illumina sequencing methods that have a combined average coverage higher than 90x. In the TPA strain Nichols genome, 134 errors were identified (25 substitutions and 109 indels), and 102 of them affected protein sequences. In the TPA SS14 genome, a total of 191 errors were identified (85 substitutions and 106 indels) and 136 of them affected protein sequences. A set of new intrastrain heterogenic regions in the TPA SS14 genome were identified including the tprD gene, where both tprD and tprD2 alleles were found. The resequenced genomes of both TPA Nichols and SS14 strains clustered more closely with related strains (i.e. strains belonging to same syphilis treponeme subcluster). At the same time, groups of Nichols-like and SS14-like strains were found to be more distantly related.

Conclusion/significance

We identified errors in 11.5% of all annotated genes and, after correction, we found a significant impact on the predicted proteomes of both Nichols and SS14 strains. Corrections of these errors resulted in protein elongations, truncations, fusions and indels in more than 11% of all annotated proteins. Moreover, it became more evident that syphilis is caused by treponemes belonging to two separate genetic subclusters.

SUBMITTER: Petrosova H

PROVIDER: S-EPMC3769245 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Resequencing of Treponema pallidum ssp. pallidum strains Nichols and SS14: correction of sequencing errors resulted in increased separation of syphilis treponeme subclusters.

Pětrošová Helena H Pospíšilová Petra P Strouhal Michal M Čejková Darina D Zobaníková Marie M Mikalová Lenka L Sodergren Erica E Weinstock George M GM Šmajs David D

PloS one 20130910 9

<h4>Background</h4>Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, is a highly clonal bacterium showing minimal genetic variability in the genome sequence of individual strains. Nevertheless, genetically characterized syphilis strains can be clearly divided into two groups, Nichols-like strains and SS14-like strains. TPA Nichols and SS14 strains were completely sequenced in 1998 and 2008, respectively. Since publication of their complete genome sequences, a number of seq ...[more]

PMID: 24058545

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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: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.

Dataset Information

Resequencing of Treponema pallidum ssp. pallidum strains Nichols and SS14: correction of sequencing errors resulted in increased separation of syphilis treponeme subclusters.

Background

Methodology/principal findings

Conclusion/significance

Publications

Resequencing of Treponema pallidum ssp. pallidum strains Nichols and SS14: correction of sequencing errors resulted in increased separation of syphilis treponeme subclusters.

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