The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.

Trichomonas vaginalis is a flagellated protozoan causing a notorious urogenital infection in humans. Due to its anaerobic metabolism, an alternative fluorescent protein that can be readily expressed in oxygen-deprived conditions is ideal. This study assessed the performance of iLOV, which does not require oxygen to function, as compared to the conventional enhanced green fluorescent protein (eGFP) in T. vaginalis. The results indicated that iLOV outperforms eGFP in both transient and stable expression, being detectable earlier and producing higher fluorescent intensity than eGFP in T. vaginalis. This finding facilitates forthcoming genetic studies that will advance the knowledge on this human parasitic infection.

Background: Trichomonas vaginalis, the causative agent of a prevalent urogenital infection in humans, is an evolutionarily divergent protozoan. Protein-coding genes in T. vaginalis are largely controlled by two core promoter elements, producing mRNAs with short 5′ UTRs. The specific mechanisms adopted by T. vaginalis to fine-tune the translation efficiency (TE) of mRNAs remain largely unknown. Results: Using both computational and experimental approaches, this study investigated two key factors influencing TE in T. vaginalis: codon usage and mRNA secondary structure. Statistical dependence between TE and codon adaptation index (CAI) highlighted the impact of codon usage on mRNA translation in T. vaginalis. A genome-wide interrogation revealed that low structural complexity at the 5′ end of mRNA followed closely by a highly structured downstream region correlates with TE variation in this organism. To validate these findings, a synthetic library of 15 synonymous iLOV genes was created, representing five mRNA folding profiles and three codon usage profiles. Fluorescence signals produced by the expression of these synonymous iLOV genes in T. vaginalis were consistent with and validated our in silico predictions. Conclusions: This study demonstrates the role of codon usage bias and mRNA secondary structure in TE of T. vaginalis mRNAs, contributing to a better understanding of the factors that influence, and possibly regulate, gene expression in this human pathogen.

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