Characterization of the chloroquine resistance transporter homologue in Toxoplasma gondii

Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein confer resistance to the antima-larial drug chloroquine. PfCRT localizes to the parasite digestive vacuole, the site of chloroquine action, where it mediates resistance by transporting chloroquine out of the digestive vacuole. PfCRT belongs to a family of transporter proteins called the chlo-roquine resistance transporter family. CRT family proteins are found throughout the Apicomplexa, in some protists, and in plants. Despite the importance of PfCRT in drug resistance, little is known about the evolution or native function of CRT proteins. The apicomplexan parasite Toxoplasma gondii contains one CRT family protein. We demonstrate that T. gondii CRT (TgCRT) colocalizes with markers for the vacuolar (VAC) compartment in these parasites. The TgCRT-containing VAC is a highly dynamic organelle, changing its morphology and protein composition between intracellular and extracellular forms of the parasite. Regulated knockdown of TgCRT expression resulted in modest reduction in parasite fitness and swelling of the VAC, indicating that TgCRT contributes to parasite growth and VAC physiology. Together, our findings provide new information on the role of CRT family proteins in apicomplexan parasites

Genetic Indicators of Drug Resistance in the Highly Repetitive Genome of Trichomonas vaginalis

Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool

Identification of a non-canonical ciliate nuclear genetic code where UAA and UAG code for different amino acids.

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel uncultured ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3'UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids

Genetic Indicators of Drug Resistance in the Highly Repetitive Genome of Trichomonas vaginalis

Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool

tRNA genes pairwise identities.

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the “universal” genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel uncultured ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3’UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.</div

Example multiple sequence alignments of <i>Plagiopyla frontata</i> genes with internal UGA codons identified by PhyloFisher with orthologous sequences spanning Eukaryota.

Example multiple sequence alignments of Plagiopyla frontata genes with internal UGA codons identified by PhyloFisher with orthologous sequences spanning Eukaryota.</p

Example tRNA Genes.

(A) Predicted secondary structure of an example tRNA-Sup(UUA) predicted to function as a lysine tRNA. The wobble position is highlighted. According to wobble-binding rules, uracil at this position can bind to either adenine or guanine in the third codon position of mRNA, allowing the suppressor tRNA to recognise both UAA and UAG stop codons. (B) Predicted secondary structure of an example tRNA-Sup(CUA) predicted to function as a glutamic acid tRNA. (C) Predicted secondary structure of the tRNA-SeC(UCA) for selenocysteine.</p

Bayesian phylogenomic analysis of 46 ciliate species and 9 outgroup Alveolata species, based on a concatenated alignment of 89 BUSCO proteins under the CAT-GTR model using PhyloBayes-MPI.

Bayesian phylogenomic analysis of 46 ciliate species and 9 outgroup Alveolata species, based on a concatenated alignment of 89 BUSCO proteins under the CAT-GTR model using PhyloBayes-MPI.</p

Genetic code prediction for Oligohymenophorea sp. PL0344.

PhyloFisher genetic code prediction for the (A) UAA and (B) UAG codons using the PhyloFisher database of 240 orthologs. Only well conserved (>70%) amino acids are considered. Colours correspond to amino acid properties and match the multiple sequence alignment in Fig 1. (C) Codetta genetic code prediction. Log decoding probabilities for the UAA and UAG codons are shown for each of the 20 standard amino acids.</p

Phylogenomic analysis of genetic code changes in the Ciliophora.

Maximum-likelihood phylogeny of 46 ciliate species and 9 outgroup species from the Alveolata, based on a concatenated alignment of 89 BUSCO proteins (40,289 amino acid sites) under the LG+F+I+R7 model using IQ-TREE. The values at branches represent statistical support from 100 non-parametric bootstraps with the LG+F+I+R7 model, 100 non-parametric bootstraps from the IQ-TREE partitioned analysis, and Bayesian posterior probabilities determined under the CAT-GTR model in PhyloBayes-MPI. Branches have full support from all three approaches (i.e., 100/100/1) except where indicated. Hyphens indicate branches that weren’t recovered under a particular analysis. Stop codon reassignments are shown (*, STOP; Q, glutamine; W, tryptophan; K, lysine; E, glutamic acid; Y, tyrosine; C, cysteine). Numbers inside solid black circles along branches indicate when a genetic code change event was inferred to have occurred (UAR = UAA and UAG). The percentage of proteins included in the concatenated alignment is shown in the bar plot, highlighting the amount of missing data per species.</p