Unraveling the role of trypanosomal ncRNA in the regulation of antigenic variation
Final Report Abstract
Populations of seemingly identical unicellular organisms show an immense level of heterogeneity with regards to morphology, growth rate and the ability to adapt to environments. For example, the ability of some bacteria, fungi and parasites to periodically switch the expression of protein isoforms on their surface, a strategy referred to as phase variation or antigenic variation, can allow pathogens to evade the host immune response and to establish lasting infections. In this process, the frequency of antigenic variation has important biological consequences. Antigen switching must occur at a rate fast enough to allow the pathogen to evade the host immune response, but not so fast that the pathogen’s antigen repertoire is exhausted before transmission to a new host has occurred. Thus, the mechanism controlling the degree of cell-to-cell heterogeneity is central for the ability of these pathogens to establish lasting infections. Yet, despite its importance, very little is known about the factors controlling cellular heterogeneity in unicellular organisms. To elucidate the mechanisms controlling cell-to-cell heterogeneity, the central goal of this research project, we studied antigenic variation in Trypanosoma brucei as a model system. T. brucei is the causative agent of sleeping sickness in humans and nagana in cattle and represents one of the best-established systems for the study of antigenic variation. More than 20 years ago it was found that the region just upstream of the actively transcribed antigen gene is crucial for its continuous expression; a short deletion resulted in activation of other antigen isoforms. Much more recently, we had found that the regions located upstream and downstream of the actively transcribed antigen gene are transcribed into long ncRNA, i.e. RNA that is not translated into proteins. The goal of the proposed work was to follow up on these findings and to determine whether the DNA regions flanking the antigen genes or the ncRNA transcribed from these regions contained elements affecting the frequency by which new antigen isoforms are activated. To be able to address these questions, we focused on the establishment of two key methods: a) a sensitive method to detect antigen switching events b) a method for the precise genome editing of the regions flanking the antigen gene. Originally, our plan was to combine immuno-lysis of cells that had not switched antigen expression with bulk RNA-seq. However, immuno-lysis of cells expressing a specific antigen isoform was either not sufficiently specific or not sufficiently efficient to evaluate switching events. Thus, we decided to establish single-cell RNA-sequencing, a method that allowed us to determine the mRNA composition present in individual parasites, yielding information on the actively transcribed antigen in T. brucei. Using single-cell RNA-sequencing, we were able to detect an increase in antigen switching following the deletion of different histone variants, demonstrating the usefulness of this approach to identify factors affecting antigen switching In addition, in an effort to establish a system allowing precise genome edits without the need to insert resistance marker genes into the genome, we established different CRISPR-Cas9 based approaches in T. brucei and demonstrated its precision in a variety of genomic contexts. Thus, while we have not yet been able to identify DNA or RNA elements affecting the antigen switching frequency, we have successfully established tools for the precise and marker-free genome editing and for the measurement of transcriptomes at the single-cell level. The development of these technologies has set the basis for future work that will hopefully soon lead to the discovery of the factors controlling the switching in antigen expression.
Publications
- (2018) Exploiting CRISPR-Cas9 technology to investigate individual histone modifications. Nucleic Acids Res, 46: e106
Vasquez, J. J., Wedel, C., Cosentino, R. O., Siegel, T. N.
(See online at https://doi.org/10.1093/nar/gky517) - (2018) Genome organization and DNA accessibility control antigenic variation in trypanosomes. Nature, 563: 121–125
Müller, L. S. M., Cosentino, R. O., Förstner, K. U., Guizetti, J., Wedel, C., Kaplan, N., Janzen, C. J., Arampatzi, P., Vogel, J., Steinbiss, S., Otto, T. D., Saliba, A. E., Sebra, R. P., Siegel, T. N.
(See online at https://doi.org/10.1038/s41586-018-0619-8)
