Universal in vitro culture model as a replacement strategy to study pathogenic treponemes

Pathogenic treponemes are bacteria causing distinct diseases such as syphilis, yaws, bejel and atypical treponematosis in humans and in some animals. Despite causing severe life-threatening infections, very little is known about their basic biology and pathogenesis. This is mainly because these bacteria are generally considered to be unculturable. Treponemes have a very small genome that lacks multiple metabolic pathways necessary for the de novo biosynthesis of various co-factors, fatty acids, and nucleotides. So, to survive and grow, these bacteria need to obtain their essential nutrients through interaction with their host cells. Therefore, a culture system that provides all of these environmental requirements should, in theory, be able to sustain the growth of these bacteria, in vitro.

However, despite rigorous attempts by the scientific community, so far, the optimisation of an axenic culture system has been unsuccessful. Therefore, for decades, large number of rabbits have been used to propagate clinically relevant treponemes. Only recently, after decades of experiments the first successful long-term cultivation of pathogenic treponemes co-cultured with rabbit skin epithelial cells (Sf1Ep) was introduced. Although technically challenging and requiring state-of-the-art technical skills, this system has opened new avenues for the study of the basic biology of pathogenic treponemes. Unfortunately, due to a very high number of treponemal cells needed to initiate the culture, the Sf1Ep co-culture system remains restricted only to rabbit-propagated strains, and cannot be used for the propagation of new strains. Moreover, even when a high number of viable treponemes are available, for unknown reasons, some treponemal strains remain unculturable under these conditions, limiting the scopes of treponemal research to only a few reference strains.
The ultimate objective of this proposal is to generate high-quality multi-omics data to systematically obtain the necessary information for the optimisation of a universal culture condition that could support the growth and propagation of multiple different treponemal strains with limited number of viable bacteria to initiate the culture. This will not only replace the use of animals for treponemal expansion and syphilis diagnosis, but will also allow us to study the basic biology of pathogenic treponemes, their pathogenesis, routes of transmission and alternative treatments.

I am aiming to using the Sf1Ep co-culture system to track the longitudinal changes in genome-wide expression patterns of treponemes during the adaptation from in vivo into the in vitro conditions. Using these transcriptional data combined with subsequent phenotypic profiling, I will model metabolic pathways and identify the metabolic interaction network of Sf1Ep cells and treponemes. Based on these observations, I will identify the essential nutrients and requirements that are crucial for the axenic culture of pathogenic treponemes. Subsequently, I will evaluate the effect of the medium supplements on the growth and viability of treponemes both in the presence and absence of Sf1Ep cells. Alternatively, I will also explore the potential of improving the co-culture system by either supplementing the Sf1Ep cells with essential nutrients or by replacing the Sf1Ep cells with other mammalian cells which may be more effective at supporting the in vitro growth of treponemes.

Our success in this project will not only pave the way for the basic science research of these fastidious bacteria, but will also significantly reduce the number of rabbits that are sacrificed for treponemal propagation and diagnosis. Furthermore, our proposed systematic pipeline could also set precedence for other similar microorganisms, translating into a vast 3Rs impact. Finally, alongside the main objectives, this project will also provide new important insights regarding the biology, physiology and genetics of pathogenic treponemes.

Very little is known about the basic biology of pathogenic treponemes, mainly as a result of our inability to propagate clinical strains under in vitro conditions. This project will build on the experience of the applicant and collaborators to generate new knowledge on key areas of treponemal biology with the aim to develop a universal in vitro culture system for these bacteria. This collaborative effort will bring together experts in Treponema biology, in vitro culture of fastidious bacteria, and metabolic-pathway bioinformatics. We will take advantage of applicants' expertise in maintaining a co-culture system with rabbit skin cells (Sf1Ep) that is currently able to support the growth of only a few treponemal strains previously propagated in experimental animals. We will comprehensively characterize the metabolic network interactions of pathogenic treponemes and Sf1Ep cells. Genomics, dual RNA-seq, experimental metabolic profiling using OmniLog, and various untargeted metabolomics approaches will be performed. These experiments will be guided by the accurately annotated Sf1Ep and treponemal reference genomes prepared as part of this project, whereby the presence of in silico predicted proteins and "putative mRNA" will be verified using Mass Spectrometry. These outputs will provide novel information about basic biology of pathogenic treponemes and will shed light on the Treponema-Sf1Ep interactions in the context of the co-culture system. In parallel, building upon our preliminary results, we will search for other cell lines capable of supporting treponemal growth. For this, we will have access to the "Cell Model Passports" database containing 1,300 different cell lines with detailed annotations. Ultimately, combining all observations, we will aim to design culture medium and condition which can universally support the growth of different species of pathogenic treponemes as a reliable replacement strategy for the use of experimental animals for treponemal expansion