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Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods.

ABSTRACT: All of our current knowledge of African trypanosome metabolism is based on results from trypanosomes grown in culture or in rodents. Drugs against sleeping sickness must however treat trypanosomes in humans. We here compare the transcriptomes of Trypanosoma brucei rhodesiense from the blood and cerebrospinal fluid of human patients with those of trypanosomes from culture and rodents. The data were aligned and analysed using new user-friendly applications designed for Kinetoplastid RNA-Seq data. The transcriptomes of trypanosomes from human blood and cerebrospinal fluid did not predict major metabolic differences that might affect drug susceptibility. Usefully, there were relatively few differences between the transcriptomes of trypanosomes from patients and those of similar trypanosomes grown in rats. Transcriptomes of monomorphic laboratory-adapted parasites grown in in vitro culture closely resembled those of the human parasites, but some differences were seen. In poly(A)-selected mRNA transcriptomes, mRNAs encoding some protein kinases and RNA-binding proteins were under-represented relative to mRNA that had not been poly(A) selected; further investigation revealed that the selection tends to result in loss of longer mRNAs.

SUBMITTER: Mulindwa J

PROVIDER: S-EPMC5842037 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods.

Mulindwa Julius J Leiss Kevin K Ibberson David D Kamanyi Marucha Kevin K Helbig Claudia C Melo do Nascimento Larissa L Silvester Eleanor E Matthews Keith K Matovu Enock E Enyaru John J Clayton Christine C

PLoS neglected tropical diseases 20180223 2

All of our current knowledge of African trypanosome metabolism is based on results from trypanosomes grown in culture or in rodents. Drugs against sleeping sickness must however treat trypanosomes in humans. We here compare the transcriptomes of Trypanosoma brucei rhodesiense from the blood and cerebrospinal fluid of human patients with those of trypanosomes from culture and rodents. The data were aligned and analysed using new user-friendly applications designed for Kinetoplastid RNA-Seq data. ...[more]

PMID: 29474390

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Project description:BackgroundHuman African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late (meningoencephalitic) stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance.Methodology/principal findingsThis was a retrospective study on a cohort of 115 T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-? synthesis were associated with stage progression and were mirrored by a reduction in TGF-? levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF.ConclusionsOur results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value.

Project description:BackgroundSleeping sickness due to Trypanosoma brucei rhodesiense has a wide spectrum of clinical presentations coupled with differences in disease progression and severity across East and Southern Africa. The disease progresses from an early (hemo-lymphatic) stage to the late (meningoencephalitic) stage characterized by presence of parasites in the central nervous system. We hypothesized that disease progression and severity of the neurological response is modulated by cytokines.MethodsA total of 55 sleeping sickness cases and 41 healthy controls were recruited passively at Lwala hospital, in Northern Uganda. A panel of six cytokines (IFN-?, IL1-?, TNF-?, IL-6, TGF-? and IL-10) were assayed from paired plasma and cerebrospinal fluid (CSF) samples. Cytokine concentrations were analyzed in relation to disease progression, clinical presentation and severity of neurological responses.ResultsMedian plasma levels (pg/ml) of IFN-? (46.3), IL-6 (61.7), TGF-? (8755) and IL-10 (256.6) were significantly higher in cases compared to controls (p< 0.0001). When early stage and late stage CSF cytokines were compared, IL-10 and IL-6 were up regulated in late stage patients and were associated with a reduction in tremors and cranioneuropathy. IL-10 had a higher staging accuracy with a sensitivity of 85.7% (95% CI, 63.7%-97%) and a specificity of 100% (95% CI, 39.8%-100%) while for IL-6, a specificity of 100% (95% CI, 47.8%-100%) gave a sensitivity of 83.3% (95% CI, 62.2%-95.3%).ConclusionOur study demonstrates the role of host inflammatory cytokines in modulating the progression and severity of neurological responses in sleeping sickness. We demonstrate here an up-regulation of IL-6 and IL-10 during the late stage with a potential as adjunct stage biomarkers. Given that both cytokines could potentially be elevated by other CNS infections, our findings should be further validated in a large cohort of patients including those with other inflammatory diseases such as cerebral malaria.

Project description:The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.

Dataset Information

Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods.

Publications

Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods.

Similar Datasets

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