Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host.

The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.

The post-transcriptional trans-acting regulator, TbZFP3, co-ordinates transmission-stage enriched mRNAs in Trypanosoma brucei.

Post-transcriptional gene regulation is essential to eukaryotic development. This is particularly emphasized in trypanosome parasites where genes are co-transcribed in polycistronic arrays but not necessarily co-regulated. The small CCCH protein, TbZFP3, has been identified as a trans-acting post-transcriptional regulator of Procyclin surface antigen expression in Trypanosoma brucei. To investigate the wider role of TbZFP3 in parasite transmission, a global analysis of associating transcripts was carried out. Examination of a subset of the selected transcripts revealed their increased abundance through mRNA stabilization upon TbZFP3 ectopic overexpression, dependent upon the integrity of the CCCH zinc finger domain. Reporter assays demonstrated that this regulation was mediated through 3'-UTR sequences for two target transcripts. Global developmental expression profiling of the cohort of TbZFP3-selected transcripts revealed their significant enrichment in transmissible stumpy forms of the parasite. This analysis of the specific mRNAs selected by the TbZFP3mRNP provides evidence for a developmental regulon with the potential to co-ordinate genes important in parasite transmission.

The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation.

Proteins with Pumilio RNA binding domains (Puf proteins) are ubiquitous in eukaryotes. Some Puf proteins bind to the 3'-untranslated regions of mRNAs, acting to repress translation and promote degradation; others are involved in ribosomal RNA maturation. The genome of Trypanosoma brucei encodes eleven Puf proteins whose function cannot be predicted by sequence analysis. We show here that epitope-tagged TbPUF7 is located in the nucleolus, and associated with a nuclear cyclophilin-like protein, TbNCP1. RNAi targeting PUF7 reduced trypanosome growth and inhibited two steps in ribosomal RNA processing.

Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei.

BACKGROUND: Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. RESULTS: In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry), thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. CONCLUSION: Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition.

The cell biology of Trypanosoma brucei differentiation.

Developmental events in the life-cycle of the sleeping sickness parasite comprise integrated changes in cell morphology, metabolism, gene expression and signalling pathways. In each case these processes differ from the eukaryotic norm. In the past three years, understanding of these developmental processes has progressed from a description of the cytological events of differentiation to a discovery of its underlying molecular controls. With an expanding set of reagents for the identification of distinct parasite life-cycle stages in the tsetse, trypanosome differentiation is being studied from the molecular to the organismal and population level. Interestingly, the new molecular discoveries provide insights into the biology of the parasite in the field.

Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei: evidence for genome-wide conservation of life-cycle stage-specific regulatory elements.

Trypanosomes represent an excellent model for the post-transcriptional regulation of gene expression because their genome is organized into polycistronic transcription units. However, few signals governing developmental stage-specific expression have been identified, with there being no compelling evidence for widespread conservation of regulatory motifs. As a tool to search for common regulatory sequences we have used the nuclear-encoded components of the cytochrome oxidase (COX) complex of the trypanosome respiratory chain. Components of this complex represent a form of post-transcriptional operon because trypanosome mitochondrial activity is unusual in being developmentally programmed. By genome analysis we identified the genes for seven components of the COX complex. Each mRNA exhibits bloodstream stage-specific instability, which is not mediated by the RNA silencing pathway but which is alleviated by cycloheximide. Reporter assays have identified regulatory regions within the 3'-untranslated regions of three COX mRNAs operating principally at the translational level, but also via mRNA stability. Interrogation of the mapped regions via oligonucleotide frequency scoring provides evidence for genome-wide conservation of regulatory sequences among a large cohort of procyclic-enriched transcripts. Analysis of the co-regulated subunits of a stage-specific enzyme is therefore a novel approach to uncover cryptic regulatory sequences controlling gene expression at the post-transcriptional level.

Phylogenetic relationships of the Wolbachia of nematodes and arthropods.

Wolbachia are well known as bacterial symbionts of arthropods, where they are reproductive parasites, but have also been described from nematode hosts, where the symbiotic interaction has features of mutualism. The majority of arthropod Wolbachia belong to clades A and B, while nematode Wolbachia mostly belong to clades C and D, but these relationships have been based on analysis of a small number of genes. To investigate the evolution and relationships of Wolbachia symbionts we have sequenced over 70 kb of the genome of wOvo, a Wolbachia from the human-parasitic nematode Onchocerca volvulus, and compared the genes identified to orthologues in other sequenced Wolbachia genomes. In comparisons of conserved local synteny, we find that wBm, from the nematode Brugia malayi, and wMel, from Drosophila melanogaster, are more similar to each other than either is to wOvo. Phylogenetic analysis of the protein-coding and ribosomal RNA genes on the sequenced fragments supports reciprocal monophyly of nematode and arthropod Wolbachia. The nematode Wolbachia did not arise from within the A clade of arthropod Wolbachia, and the root of the Wolbachia clade lies between the nematode and arthropod symbionts. Using the wOvo sequence, we identified a lateral transfer event whereby segments of the Wolbachia genome were inserted into the Onchocerca nuclear genome. This event predated the separation of the human parasite O. volvulus from its cattle-parasitic sister species, O. ochengi. The long association between filarial nematodes and Wolbachia symbionts may permit more frequent genetic exchange between their genomes.

Identification and stage-specific association with the translational apparatus of TbZFP3, a CCCH protein that promotes trypanosome life-cycle development.

The post-transcriptional control of gene expression is becoming increasingly important in the understanding of regulated events in eukaryotic cells. The parasitic kinetoplastids have a unique reliance on such processes, because their genome is organized into polycistronic transcription units in which adjacent genes are not coordinately regulated. Indeed, the number of RNA-binding proteins predicted to be encoded in the genome of kinetoplastids is unusually large, invoking the presence of unique RNA regulators dedicated to gene expression in these evolutionarily ancient organisms. Here, we report that a small CCCH zinc finger protein, TbZFP3, enhances development between life-cycle stages in Trypanosoma brucei. Moreover, we demonstrate that this protein interacts both with the translational machinery and with other small CCCH proteins previously implicated in trypanosome developmental control. Antibodies to this protein also co-immunoprecipitate EP procyclin mRNA and encode the major surface antigen of insect forms of T. brucei. Strikingly, although TbZFP3 is constitutively expressed, it exhibits developmentally regulated association with polyribosomes, and mutational analysis demonstrates that this association is essential for the expression of phenotype. TbZFP3 is therefore a novel regulator of developmental events in kinetoplastids that acts at the level of the post-transcriptional control of gene expression.

Wolbachia genomes: revealing the biology of parasitism and mutualism.

Wolbachia bacteria are endosymbiotic partners of many animal species, in which they behave as either parasites (in arthropod hosts) or mutualists (in nematode hosts). What biochemistry and biology underpin these diverse lifestyles? The recent complete sequencing of genomes from Wolbachia that infect the arthropod Drosophila melanogaster and the nematode Brugia malayi, together with the partial genome sequencing of three Wolbachia strains found in drosophilids, enables this question to begin to be addressed. Parasitic arthropod Wolbachia are characterized by the presence of phages that carry ankyrin-repeat proteins; these proteins might be exported to the host cell to manipulate reproduction. In nematode Wolbachia, which lack these phages, several biochemical pathways can deliver essential metabolites to the nematode hosts. Nematode Wolbachia might also have a role in modulating the mammalian host immune system but the sequenced Wolbachia genomes lack the genes to synthesize lipopolysaccharide, raising questions about the nature of the inducing molecule. The Wolbachia surface protein might carry out this function.

Most of the response elicited against Wolbachia surface protein in filarial nematode infection is due to the infective larval stage.

Immune responses to the intracellular Wolbachia bacteria of filarial nematodes are thought to contribute to the pathologic process of filarial infection. Here, we compare antibody responses of subjects living in an area where lymphatic filariasis is endemic with antibody responses elicited in a murine model of filarial infection, to provide evidence that the infective larval stage (L3), not adult nematodes, are the primary inducer of responses against Wolbachia. In human subjects, antibody responses to Brugia malayi Wolbachia surface protein (WSP) are most often correlated with antibody responses to the L3 stage of B. malayi. Analysis of anti-WSP responses induced in mice by different stages of the rodent filariae Litomosoides sigmodontis shows that the strongest anti-WSP response is elicited by the L3 stage. Although adult filarial nematode death may play a role in the generation of an anti-WSP response, it is the L3 stage that is the major source of immunogenic material, and incoming L3 provide a continual boosting of the anti-WSP response. Significant exposure to the endosymbiotic bacteria may occur earlier in nematode infection than previously thought, and the level of exposure to infective insect bites may be a key determinant of disease progression.

High throughput immuno-screening of cDNA expression libraries produced by in vitro recombination; exploring the Plasmodium falciparum proteome.

Improved Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo-capping with in vitro recombination and directional cloning of cDNA inserts into a plasmid vector that expresses sequences as thioredoxin fusion proteins. A novel procedure has also been developed for the rapid identification of seropositive clones on high-density filters, using direct labelling of P. falciparum immune immunoglobulin with fluorescein isothiocynate (FITC). This approach combines the advantages of recombination-assisted cDNA cloning with high throughput, non-radioactive serological screening of expression libraries. Production of replicate colony matrices allows the identification of antigens recognised by different pools with different specificities from residents of a malaria endemic region. Analyses of DNA sequences derived from sero-reactive colonies indicate that this is an effective method for producing recombinant proteins that react with antibodies from malaria-exposed individuals. This approach permits the systematic construction of a database of antigenic proteins recognised by sera from malaria-exposed individuals.

Are filarial nematode Wolbachia obligate mutualist symbionts?

The intracellular symbiotic bacteria of filarial nematodes have inspired new ideas for the control of disease using antibacterial drugs. For effective, long-term control, this requires that the bacteria are essential to their nematode hosts. Two recent studies offer conflicting evidence: long, close coevolution between most filarial nematodes and their symbionts contrasts with many species having naturally lost them. An attempt to transfer symbionts to an uninfected host found that the bacteria did not thrive, suggesting they are adapted to one host.