Abstract:
Trichomonas vaginalis is the agent of one of the most common sexually transmitted infections worldwide, known as trichomoniasis There is a lack of attention on this infectious organism which remains understudied. More than just a sexually transmitted pathogen, T. vaginalis is a deep-branching protozoan, an interesting model for studying early evolution of eukaryotes. Compared to other single cell eukaryotes, T. vaginalis has an unusually large genome (160 MB) with one of the highest coding capabilities (~60,000 protein-coding genes). Further attention is needed to study its genetic regulatory mechanisms.
Among these mechanisms, RNA interference (RNAi) is thought to have evolved in all eukaryotes but the appearance of miRNAs remains disputable. RNAi can drastically reduce the expression of a gene without removing it from the genome and has become a valuable tool for studying gene function. Evidence for RNAi activities has been documented in deep-branching protozoa, including Giardia lamblia, Entamoeba histolytica and Trypanosoma brucei. For that latter, RNAi has been a tool of choice for genetic ablation. In T. vaginalis, there has been sporadic reports on repression of gene expression by double stranded RNAs. It was only recently demonstrated that the former PhD student Shuqi (Edward) Wang in our laboratory demonstrated that constitutive transcription of short hairpin RNAs caused repression of a reporter gene
Here, I wanted to evaluate the potential of RNAi in T. vaginalis. To do that I needed to understand the basic components of this machinery and their individual characteristics. Firstly, I revisited T. vaginalis genome to carry out a bioinformatics survey on the possible protein components of this machinery. In comparison to other eukaryotes, as predicted by the initial genome annotation, I confirmed the existence of three homologues: one Dicer (TvDicer) and two Argonautes (TvAgo1 and TvAgo2). Upon examining alignments and domains, I found that these homologues have the minimal features that are expected from functional RNAi proteins. I detected evidence for their expression by examining RNA-seq datasets.
To characterize these protein components of T. vaginalis RNAi, I attempted to express the recombinant proteins utilizing heterologous expression systems in Escherichia coli. Unfortunately, solubility became a hurdle for obtaining significant yields of the
recombinant proteins. After many methodological variations on the prokaryotic expression system, I managed to obtain TvDicer (RNase IIIb) and TvAgo2 in soluble form. Adjustments to the protocol will be needed to evaluate if yields will be sufficient for downstream structural and functional experiments.
Finally, to provide solid evidence of RNAi activities in T. vaginalis, I performed gene knockdown. Firstly, I confirmed Shuqi’s result on the knockdown of the episomal iLOV using digital droplet PCR. Then, I designed multiple shRNA to knockdown the expression of four endogenous genes (i.e. in their chromosomal content) for the first time in this organism. I showed that we could downregulate the expression of these genes efficiently, by up to 79%. Additionally, I observed that expression inhibition could be enhanced by co-expressing the shRNA with TvAgo2. Overall, my work supported that RNAi is functional in T. vaginalis, can be activated by producing exogenous shRNAs within cells and thus might be a valuable tool for studying gene function in this organism.