The languages of parasite communication

Although it is regarded as self-evident that parasites interact with their hosts, with the primary aim of enhancing their own survival and transmission, the extent to which unicellular parasites communicate with each has been severely underestimated. Recent publications show that information is commonly exchanged between parasites of the same species and that this can govern their decisions to divide, to differentiate or to migrate as a group. Communication can take the form of soluble secreted factors, extra- cellular vesicles or contact between cells. Extracellular parasites can do this directly, while intracellular parasites use the infected host cell – or components derived from it – as an intermediary. By emitting signals that can be dispersed within the host, parasites can also have long-distance effects on the course of an infection and its pathology. This article presents an overview of recent developments in this field and draws attention to some older work that merits re-examination

Bidirectional silencing of RNA polymerase I transcription by a strand switch region in Trypanosoma brucei

The procyclin genes in Trypanosoma brucei are transcribed by RNA polymerase I as part of 5-10 kb long polycistronic transcription units on chromosomes VI and X. Each procyclin locus begins with two procyclin genes followed by at least one procyclin-associated gene (PAG). In procyclic (insect midgut) form trypanosomes, PAG mRNA levels are about 100-fold lower than those of procyclins. We show that deletion of PAG1, PAG2 or PAG3 results in increased mRNA levels from downstream genes in the same transcription unit. Nascent RNA analysis revealed that most of the effects are due to increased transcription elongation in the knockouts. Furthermore, transient and stable transfections showed that sequence elements on both strands of PAG1 can inhibit Pol I transcription. Finally, by database mining we identified 30 additional PAG-related sequences that are located almost exclusively at strand switch regions and/or at sites where a change of RNA polymerase type is likely to occu

The RNA export factor TbMex67 connects transcription and RNA export in Trypanosoma brucei and sets boundaries for RNA polymerase I.

TbMex67 is the major mRNA export factor known to date in trypanosomes, forming part of the docking platform within the nuclear pore. To explore its role in co-transcriptional mRNA export, recently reported in Trypanosoma brucei, pulse labelling of nascent RNAs with 5-ethynyl uridine (5-EU) was performed with cells depleted of TbMex67 and complemented with a dominant-negative mutant (TbMex67-DN). RNA polymerase (Pol) II transcription was unaffected, but the procyclin loci, which encode mRNAs transcribed by Pol I from internal sites on chromosomes 6 and 10, showed increased levels of 5-EU incorporation. This was due to Pol I readthrough transcription, which proceeded beyond the procyclin and procyclin-associated genes up to the Pol II transcription start site on the opposite strand. Complementation by TbMex67-DN also increased Pol I-dependent formation of R-loops and γ-histone 2A foci. The DN mutant exhibited reduced nuclear localisation and binding to chromatin compared to wild-type TbMex67. Together with its interaction with chromatin remodelling factor TbRRM1 and Pol II, and transcription-dependent association of Pol II with nucleoporins, our findings support a role for TbMex67 in connecting transcription and export in T. brucei. In addition, TbMex67 stalls readthrough by Pol I in specific contexts, thereby limiting R-loop formation and replication stress

Persistence of Trypanosoma brucei as early procyclic forms and social motility are dependent on glycosylphosphatidylinositol transamidase.

Glycosylphosphatidylinositol (GPI)-linked molecules are surface-exposed membrane components that influence the infectivity, virulence and transmission of many eukaryotic pathogens. Procyclic (insect midgut) forms of Trypanosoma brucei do not require GPI-anchored proteins for growth in suspension culture. Deletion of TbGPI8, and inactivation of the GPI:protein transamidase complex, is tolerated by cultured procyclic forms. Using a conditional knockout, we show TbGPI8 is required for social motility (SoMo). This collective migration by cultured early procyclic forms has been linked to colonization of the tsetse fly digestive tract. The SoMo-negative phenotype was observed after a lag phase with respect to loss of TbGPI8 and correlated with an unexpectedly slow loss of procyclins, the major GPI-anchored proteins. Procyclins are not essential for SoMo, however, suggesting a requirement for at least one other GPI-anchored protein. Loss of TbGPI8 initiates the transition from early to late procyclic forms; this effect was observed in a subpopulation in suspension culture, and was more pronounced when cells were cultured on SoMo plates. Our results indicate two, potentially interlinked, scenarios that may explain the previously reported failure of TbGPI8 deletion mutants to establish a midgut infection in the tsetse fly: interference with stage-specific gene expression and absence of SoMo

Developmental dynamics of mitochondrial mRNA abundance and editing reveal roles for temperature and the differentiation-repressive kinase RDK1 in cytochrome oxidase subunit II mRNA editing.

Developmental regulation of mitochondrial uridine insertion/deletion editing in Trypanosoma brucei is necessary to modulate parasite metabolism as it shifts from dependence on glycolysis for ATP production in the mammalian bloodstream form (BSF) to oxidative phosphorylation in the insect procyclic form (PCF). However, the timing and stimuli that regulate mRNA editing have been poorly characterized. Here, we utilized a pleomorphic T. brucei strain and quantitative RT-PCR and droplet digital PCR analyses to evaluate the changes in total mRNA abundance and editing as parasites progressively differentiate from slender BSF to PCF and investigate the effect of individual stimuli on mitochondrial gene expression. We observed little change during the slender-to-stumpy BSF transition. Rather, we found that mainly the mitochondrial cytochrome (COI, COII, COIII, and CYb) mRNAs are upregulated within 24 h after stumpy BSF is stimulated to differentiate to PCF in vitro and during in vivo tsetse fly infections. Temperature reduction from 37°C to 27°C is a critical factor for increasing the editing of COII and COIII mRNAs and COIV protein expression but not the editing of CYb mRNA or RISP protein expression. We further demonstrate that the depletion of the differentiation-repressive kinase RDK1 couples with temperature reduction to stimulate COII mRNA editing, and the accessory factor p22 is required for the cold-responsive upregulation of COII mRNA editing. Overall, we show that cytochrome mRNAs are regulated during development by distinct stimuli through a variety of methods to increase their abundance and/or editing. IMPORTANCE Trypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock. The parasite has a complex life cycle consisting of several developmental forms in the human and tsetse fly insect vector. Both the mammalian and insect hosts provide different nutritional environments, so T. brucei must adapt its metabolism to promote its survival and to complete its life cycle. As T. brucei is transmitted from the human host to the fly, the parasite must regulate its mitochondrial gene expression through a process called uridine insertion/deletion editing to achieve mRNAs capable of being translated into functional respiratory chain proteins required for energy production in the insect host. Therefore, it is essential to understand the mechanisms by which T. brucei regulates mitochondrial gene expression during transmission from the mammalian host to the insect vector

Nutrient availability regulates proline/alanine transporters in Trypanosoma brucei

Trypanosoma brucei is a species of unicellular parasite that can cause severe diseases in livestock and humans, including African trypanosomiasis and Chagas disease. Adaptation to diverse environments and changes in nutritional conditions is essential for T. brucei to establish an infection when changing hosts or during invasion of different host tissues. One such adaptation is the ability of T. brucei to rapidly switch its energy metabolism from glucose metabolism in the mammalian blood to proline catabolism in the insect stages and vice versa. However, the mechanisms that support the parasite's response to nutrient availability remain unclear. Using RNAseq and qRT-PCR, we investigated the response of T. brucei to amino acid or glucose starvation and found increased mRNA levels of several amino acid transporters, including all genes of the amino acid transporter AAT7-B subgroup. Functional characterization revealed that AAT7-B members are plasma membrane-localized in T. brucei and when expressed in Saccharomyces cerevisiae supported the uptake of proline, alanine, and cysteine, while other amino acids were poorly recognized. All AAT7-B members showed a preference for proline, which is transported with high or low affinity. RNAimediated AAT7-B downregulation resulted in a reduction of intracellular proline concentrations and growth arrest under low proline availability in cultured procyclic form parasites. Taken together, these results suggest a role of AAT7-B transporters in the response of T. brucei to proline starvation and proline catabolism

tRNASec is transcribed by RNA polymerase II in Trypanosoma brucei but not in humans

Nuclear-encoded tRNAs are universally transcribed by RNA polymerase III (Pol-III) and contain intragenic promoters. Transcription of vertebrate tRNASec however requires extragenic promoters similar to Pol-III transcribed U6 snRNA. Here, we present a comparative analysis of tRNASec transcription in humans and the parasitic protozoa Trypanosoma brucei, two evolutionary highly diverged eukaryotes. RNAi-mediated ablation of Pol-II and Pol-III as well as oligo-dT induced transcription termination show that the human tRNASec is a Pol-III transcript. In T. brucei protein-coding genes are polycistronically transcribed by Pol-II and processed by trans-splicing and polyadenylation. tRNA genes are generally clustered in between polycistrons. However, the trypanosomal tRNASec genes are embedded within a polycistron. Their transcription is sensitive to α-amanitin and RNAi-mediated ablation of Pol-II, but not of Pol-III. Ectopic expression of the tRNASec outside but not inside a polycistron requires an added external promoter. These experiments demonstrate that trypanosomal tRNASec, in contrast to its human counterpart, is transcribed by Pol-II. Synteny analysis shows that in trypanosomatids the tRNASec gene can be found in two different polycistrons, suggesting that it has evolved twice independently. Moreover, intron-encoded tRNAs are present in a number of eukaryotic genomes indicating that Pol-II transcription of tRNAs may not be restricted to trypanosomatid

NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway

Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5′UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA expor