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Transcriptomes of newly-isolated Trypanosoma brucei rhodesiense reveal hundreds of mRNAs that are co-regulated with stumpy-form markers.

Mulindwa J, Mercé C, Matovu E, Enyaru J, Clayton C - BMC Genomics (2015)

Bottom Line: Numerous mRNAs encoding proteins of no known function were either increased or decreased.Many of the mRNAs that were decreased in cells with elevated PIP39 reflected reduced cell division.These transcriptomes suggest new avenues for research into the regulation of trypanosome differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Sports Science, College of Natural Sciences, Makerere University, P.O.Box 7062, Kampala, Uganda. mulindwajul@yahoo.com.

ABSTRACT

Background: During natural Trypanosoma brucei infections, the parasites differentiate spontaneously into a non-dividing "stumpy" form when a certain level of parasitaemia is attained. This form is metabolically adapted for rapid further differentiation into procyclic forms upon uptake by Tsetse flies.

Results: We describe here four central Ugandan isolates of Trypanosoma brucei rhodesiense that have undergone only three rodent passages since isolation from human patients. As expected, SNP analysis shows that these isolates are more closely related to each other than to the commonly used strains Lister 427, Antat1.1, and TREU927. TREU927 generally has smaller copy numbers of repeated genes than the other strains, while Lister 427 trypanosomes with a 30-year history of in vitro culture and cloning have more histone genes than the other isolates. The recently isolated trypanosomes were grown in rats, and their transcriptomes characterised. In comparison with cultured procyclic and bloodstream forms, there were increases in mRNAs encoding the stumpy-form markers ESAG9 and PIP39, with coordinated alterations in the levels of over 600 additional mRNAs. Numerous mRNAs encoding proteins of no known function were either increased or decreased. The products of the mRNAs that were increased in parallel with PIP39 included not only enzymes of procyclic-form metabolism, but also components of the translational and RNA control machineries. Many of the mRNAs that were decreased in cells with elevated PIP39 reflected reduced cell division.

Conclusions: These transcriptomes suggest new avenues for research into the regulation of trypanosome differentiation.

No MeSH data available.


Related in: MedlinePlus

Comparison of transcriptomes of pleomorphic bloodstream forms with laboratory-adapted trypanosomes. a Number of genes showing differences in expression, relative to cultured bloodstream-form Lister 427 parasites at 1 × 106 /mL. BT: data from three rat infections as described in [23]. PC: Procyclic-form Lister 427 parasites at no more than 2.5 × 106 /mL. All results are for poly(A)+ mRNA, and only one representative of each gene family is included. We selected genes with at least 3-fold regulation, and an adjusted p-value threshold of 0.001. b Relative levels of mRNA (reads per million reads) from PIP39 and the ESAG9 locus Tb 927.9.7370 in the different samples
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Fig3: Comparison of transcriptomes of pleomorphic bloodstream forms with laboratory-adapted trypanosomes. a Number of genes showing differences in expression, relative to cultured bloodstream-form Lister 427 parasites at 1 × 106 /mL. BT: data from three rat infections as described in [23]. PC: Procyclic-form Lister 427 parasites at no more than 2.5 × 106 /mL. All results are for poly(A)+ mRNA, and only one representative of each gene family is included. We selected genes with at least 3-fold regulation, and an adjusted p-value threshold of 0.001. b Relative levels of mRNA (reads per million reads) from PIP39 and the ESAG9 locus Tb 927.9.7370 in the different samples

Mentions: We next obtained transcriptomes from the four isolates. Each was used to infect two rats, and the parasites were allowed to grow for 7 days, by which time the parasitaemias were estimated at between 2 × 108/ml and 6 × 108/ml [17]. We prepared RNA from buffy-coat parasites with minimal handling, since purification changes trypanosome transcriptomes [23]. Replicate RNA samples were analysed by high-throughput cDNA sequencing (RNASeq). Approximately 70 % of the reads aligned to the trypanosome genome (12–30 million reads). Initial analysis showed that the transcriptomes of replicates correlated best with each other (Additional file 4: Table S3, sheet 1). We compared them with previously published results for cultured T. b. brucei Lister 427 bloodstream forms and procyclic forms [24–27], and for lab-adapted T. b. rhodesiense (strain Tbr729) grown in rats [23]. Even when strict criteria were applied (Padj <0.001, >3-fold difference in expression) (Additional file 5: Table S4), the numbers of “significantly regulated” genes in the new isolate transcriptomes were striking (Fig. 3a). Notably, there was clear up-regulation of the PIP39 mRNA, especially in the LW032 and LW042 samples, but also to some extent in the others (Fig. 3b, Additional file 4: Table S3, sheet 5). In addition, there were increases in reads that mapped to ESAG9 genes, although the pattern was more heterogeneous (Fig. 3b), presumably due to inter-strain differences in ESAG9 gene content, as well as use of different VSG expression sites (which contain ESAG9 genes) (Additional file 4: Table S3, sheet 5). PAD1 is a member of a multi-gene family. Read counts for the PAD family as a whole were increased in LW032 and LW042 transcriptomes (Additional file 4: Table S3, sheet 5) but we cannot tell which paralogue they came from. These expression profiles suggested that the trypanosome samples from blood contained mixtures of trypanosomes at various stages of stumpy-form differentiation, with least stumpy-form expression in the Tbr729 sample, more in LW031 and LW024, and most in LW032 and LW042. The mRNAs that were most strongly regulated in LW032 and LW042 may therefore be stumpy-specific. This designation is however very tentative: although the parasitaemias in all rats were comparable with those seen at the first peak in mice, they were not counted accurately and we did not assess trypanosome morphology before RNA preparation.Fig. 3


Transcriptomes of newly-isolated Trypanosoma brucei rhodesiense reveal hundreds of mRNAs that are co-regulated with stumpy-form markers.

Mulindwa J, Mercé C, Matovu E, Enyaru J, Clayton C - BMC Genomics (2015)

Comparison of transcriptomes of pleomorphic bloodstream forms with laboratory-adapted trypanosomes. a Number of genes showing differences in expression, relative to cultured bloodstream-form Lister 427 parasites at 1 × 106 /mL. BT: data from three rat infections as described in [23]. PC: Procyclic-form Lister 427 parasites at no more than 2.5 × 106 /mL. All results are for poly(A)+ mRNA, and only one representative of each gene family is included. We selected genes with at least 3-fold regulation, and an adjusted p-value threshold of 0.001. b Relative levels of mRNA (reads per million reads) from PIP39 and the ESAG9 locus Tb 927.9.7370 in the different samples
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Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4696300&req=5

Fig3: Comparison of transcriptomes of pleomorphic bloodstream forms with laboratory-adapted trypanosomes. a Number of genes showing differences in expression, relative to cultured bloodstream-form Lister 427 parasites at 1 × 106 /mL. BT: data from three rat infections as described in [23]. PC: Procyclic-form Lister 427 parasites at no more than 2.5 × 106 /mL. All results are for poly(A)+ mRNA, and only one representative of each gene family is included. We selected genes with at least 3-fold regulation, and an adjusted p-value threshold of 0.001. b Relative levels of mRNA (reads per million reads) from PIP39 and the ESAG9 locus Tb 927.9.7370 in the different samples
Mentions: We next obtained transcriptomes from the four isolates. Each was used to infect two rats, and the parasites were allowed to grow for 7 days, by which time the parasitaemias were estimated at between 2 × 108/ml and 6 × 108/ml [17]. We prepared RNA from buffy-coat parasites with minimal handling, since purification changes trypanosome transcriptomes [23]. Replicate RNA samples were analysed by high-throughput cDNA sequencing (RNASeq). Approximately 70 % of the reads aligned to the trypanosome genome (12–30 million reads). Initial analysis showed that the transcriptomes of replicates correlated best with each other (Additional file 4: Table S3, sheet 1). We compared them with previously published results for cultured T. b. brucei Lister 427 bloodstream forms and procyclic forms [24–27], and for lab-adapted T. b. rhodesiense (strain Tbr729) grown in rats [23]. Even when strict criteria were applied (Padj <0.001, >3-fold difference in expression) (Additional file 5: Table S4), the numbers of “significantly regulated” genes in the new isolate transcriptomes were striking (Fig. 3a). Notably, there was clear up-regulation of the PIP39 mRNA, especially in the LW032 and LW042 samples, but also to some extent in the others (Fig. 3b, Additional file 4: Table S3, sheet 5). In addition, there were increases in reads that mapped to ESAG9 genes, although the pattern was more heterogeneous (Fig. 3b), presumably due to inter-strain differences in ESAG9 gene content, as well as use of different VSG expression sites (which contain ESAG9 genes) (Additional file 4: Table S3, sheet 5). PAD1 is a member of a multi-gene family. Read counts for the PAD family as a whole were increased in LW032 and LW042 transcriptomes (Additional file 4: Table S3, sheet 5) but we cannot tell which paralogue they came from. These expression profiles suggested that the trypanosome samples from blood contained mixtures of trypanosomes at various stages of stumpy-form differentiation, with least stumpy-form expression in the Tbr729 sample, more in LW031 and LW024, and most in LW032 and LW042. The mRNAs that were most strongly regulated in LW032 and LW042 may therefore be stumpy-specific. This designation is however very tentative: although the parasitaemias in all rats were comparable with those seen at the first peak in mice, they were not counted accurately and we did not assess trypanosome morphology before RNA preparation.Fig. 3

Bottom Line: Numerous mRNAs encoding proteins of no known function were either increased or decreased.Many of the mRNAs that were decreased in cells with elevated PIP39 reflected reduced cell division.These transcriptomes suggest new avenues for research into the regulation of trypanosome differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Sports Science, College of Natural Sciences, Makerere University, P.O.Box 7062, Kampala, Uganda. mulindwajul@yahoo.com.

ABSTRACT

Background: During natural Trypanosoma brucei infections, the parasites differentiate spontaneously into a non-dividing "stumpy" form when a certain level of parasitaemia is attained. This form is metabolically adapted for rapid further differentiation into procyclic forms upon uptake by Tsetse flies.

Results: We describe here four central Ugandan isolates of Trypanosoma brucei rhodesiense that have undergone only three rodent passages since isolation from human patients. As expected, SNP analysis shows that these isolates are more closely related to each other than to the commonly used strains Lister 427, Antat1.1, and TREU927. TREU927 generally has smaller copy numbers of repeated genes than the other strains, while Lister 427 trypanosomes with a 30-year history of in vitro culture and cloning have more histone genes than the other isolates. The recently isolated trypanosomes were grown in rats, and their transcriptomes characterised. In comparison with cultured procyclic and bloodstream forms, there were increases in mRNAs encoding the stumpy-form markers ESAG9 and PIP39, with coordinated alterations in the levels of over 600 additional mRNAs. Numerous mRNAs encoding proteins of no known function were either increased or decreased. The products of the mRNAs that were increased in parallel with PIP39 included not only enzymes of procyclic-form metabolism, but also components of the translational and RNA control machineries. Many of the mRNAs that were decreased in cells with elevated PIP39 reflected reduced cell division.

Conclusions: These transcriptomes suggest new avenues for research into the regulation of trypanosome differentiation.

No MeSH data available.


Related in: MedlinePlus