Comparative transcriptomics of phylogenetically selected pathogenic treponemes cultivated in vitro under different conditions: First insight to the expression changes linked with genomic variants

Project Information

ComTransTrep

Grant agreement ID: 895136

DOI

10.3030/895136

Project closed

EC signature date 23 April 2020

Start date 1 February 2021

End date 4 June 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

GENOME RESEARCH LIMITED LBG
United Kingdom

Periodic Reporting for period 1 - ComTransTrep (Comparative transcriptomics of phylogenetically selected pathogenic treponemes cultivated in vitro under different conditions: First insight to the expression changes linked with genomic variants)

Reporting period: 2021-02-01 to 2023-01-31

Summary of the context and overall objectives of the project

Despite over a century of scientific research on Treponema pallidum subsp. pallidum (TPA), the infectious agent of syphilis, our understanding of its biology is very limited. This is mainly because, in general, pathogenic treponemes are considered uncultivable bacteria, and the very small number of treponemal cells that can be obtained from clinical samples are insufficient for most scientific experiments.

In 2018, a researcher reported the first successful long-term in vitro co-cultivation of TPA with rabbit skin epithelial cells (Sf1Ep) in TPCM-2 medium. Despite being technically challenging, requiring specially trained personnel, and being limited to strains previously propagated in experimental animals, this culture system holds the potential to enhance our understanding of treponemal biology.

The main aims of the ComTransTrep project were to take advantage of this co-culture system, revise the transcriptional landscape of TPA, and provide a fundamental understanding of how different TPA strains vary at the transcriptomic level. Furthermore, I intended to compare these data with the genomes, thereby creating an opportunity to link genomic data to functional transcriptomics and, ultimately, to phenotype.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

With support from inventors of the co-culture system and other leading scientist in the field, I managed to establish this culture system at Wellcome Sanger Institute (WSI). This makes the WSI one of the few research institutes in the world with this capacity; and the first laboratory in the UK to establish this system. To date, very limited transcriptional data from TPA has been reported. This is likely due to the challenges that are associated with extraction and purification of high-quality RNA from these bacteria. I tackled this issue by specifically optimising an RNA extraction protocol and library preparation workflow, which enabled me to obtain the first high-quality transcriptome of TPA. Subsequently, I have created a pipeline for the directional RNA-seq of the in vitro cultured treponemes, including specific steps for RNA extraction, pre-library treatments, library preparation and bioinformatics analyses.

Based on the evidence from our high-quality transcriptomics data, we were able to confirm transcriptional landscape of TPA. Furthermore, we identified which regions are likely miss-annotated and corrected existing coding sequence (CDS) predictions in the latest available TPA reference. Moreover, we were able to identify an abundance of anti-sense RNAs as well as various transcripts arising from intergenic regions. In fact, more than half of the transcripts that have been identified to be in conflict with the current annotation were matched with alternative CDSs. These observations are fundamental to our understanding of Treponema biology and warrant further investigation to clarify the role of these RNA molecules.

To raise public awareness about syphilis and the challenges of TPA research, a collaboration with bioartist Anna Dimitru was established. This partnership birthed the "Syphilis dress" project, which has been showcased in several exhibitions and seminars. More details can be foudn in the provided URL address.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We have addressed a universal problem in biology: the lack of experimental validation for in silico gene predictions. By reconstructing a high-quality transcriptome profile of TPA, I have provided a more accurate representation of gene transcription, which is essential for understanding the functional elements of the genome. This innovative approach has revealed that most annotated regions, including those for hypothetical proteins, are transcribed, suggesting their potential importance in bacterial survival. Moreover, the discovery of abundant asRNA across the TPA genome and the identification of transcripts not present in the original annotation can lead to the development of new diagnostic tools or therapeutic targets.

Scanning Electron Microscopy of Treponema pallidum (David Goulding, Wellcome Sanger Institute, UK)