Christine Clayton

Life without transcriptional control? From fly to man and back again.

All organisms adapt to changes in their environment by adjustments in gene expression, and in all organisms, from Escherichia coli to man, the most important control point is at transcription initiation. All, that is, except those belonging to one very small family of early‐branching eukaryotes, which seems to have completely lost the ability to regulate transcription by RNA polymerase II. The organisms concerned are unicellular, spindle‐ like flagellates that flourish in the digestive systems of arthropods, in the blood, macrophages and brains of vertebrates from humans to lizards, and even in the sap of coconut palms and lemon trees. Many of them are able to multiply both in a vertebrate (or plant) and an invertebrate, which serves to transmit the parasites from one vertebrate (or plant) to the next. Adaptation to the two distinct environments, with different temperatures, nutrients and defences, requires major changes in gene expression. Yet this seems to be achieved in the total absence of any developmental regulation of RNA polymerase II; perhaps even without any specific polymerase II transcription initiation. This extraordinary state of affairs might be written off as an irrelevant evolutionary quirk (and, indeed, might have even gone unnoticed) if it were not for the fact that some of the organisms concerned, the trypanosomes and the leishmanias, kill millions of people every year (http://www.who.ch). The leishmanias cause a spectrum of diseases ranging from self‐resolving skin ulcers to lethal infection of the internal organs. One‐and‐a‐half to two million people are newly infected every year in the tropics and southern Europe. Leishmania has an extracellular form in the gut of the vector, the sand‐fly, but multiply as spherical aflagellate forms within the lysosomes of mammalian macrophages. Leishmania must be phagocytosed without activating the host macrophage and must combat oxidative, acidic and proteolytic stresses. The South …

Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress

In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione–glutathione reductase system found in most other organisms. To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline‐inducible promoter. This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild‐type levels by adjusting the concentration of added tetracycline. In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild‐type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant. In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC50 = 3.5 μM) was observed compared with the wild type (EC50 = 223 μM). The depletion of TRYR had no effect on susceptibility to melarsen oxide. The infectivity and virulence of the parasites in mice was dependent upon tetracycline‐regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed.

The exosome of Trypanosoma brucei

The yeast exosome is a complex of at least 10 essential 3′–5′ riboexonucleases which is involved in 3′‐processing of many RNA species. An exosome‐like complex has been found or predicted to exist in other eukaryotes but not in Escherichia coli. The unicellular parasite Trypanosoma brucei diverged very early in eukaryotic evolution. We show here that T.brucei contains at least eight exosome subunit homologs, but only a subset of these associate in a complex. Accordingly, the T.brucei exosome is smaller than that of yeast. Both free and complex‐associated homologs are essential for cell viability and are involved in 5.8S rRNA maturation. We suggest that the exosome was present in primitive eukaryotes, and became increasingly complex during subsequent evolution.

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant‐specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome‐enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5′ ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha‐amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.

Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei

Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is metabolized to pyruvate. Aerobic metabolism depends on the activities of glycosomal triosephosphate isomerase and a mitochondrial glycerophosphate oxidase, and on glycerophosphate<-->dihydroxyacetone phosphate exchange across the glycosomal membrane. Using a combination of genetics and computer modelling, we show that triosephosphate isomerase is probably essential for bloodstream trypanosome survival, but not for the insect-dwelling procyclics, which preferentially use amino acids as an energy source. When the enzyme level decreased to about 15% of that of the wild-type, the growth rate was halved. Below this level, a lethal rise in dihydroxyacetone phosphate was predicted. Expression of cytosolic triosephosphate isomerase inhibited cell growth. Attempts to knockout the trypanosome alternative oxidase genes (which are needed for glycerophosphate oxidase activity) were unsuccessful, but when we lowered the level of the corresponding mRNA by expressing a homologous double-stranded RNA, oxygen consumption was reduced fourfold and the rate of trypanosome growth was halved.

Heat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2α phosphorylation at Thr169

In trypanosomes there is an almost total reliance on post-transcriptional mechanisms to alter gene expression; here, heat shock was used to investigate the response to an environmental signal. Heat shock rapidly and reversibly induced a decrease in polysome abundance, and the consequent changes in mRNA metabolism were studied. Both heat shock and polysome dissociation were necessary for (1) a reduction in mRNA levels that was more rapid than normal turnover, (2) an increased number of P-body-like granules that contained DHH1, SCD6 and XRNA, (3) the formation of stress granules that remained largely separate from the P-body-like granules and localise to the periphery of the cell and, (4) an increase in the size of a novel focus located at the posterior pole of the cell that contain XRNA, but neither DHH1 nor SCD6. The response differed from mammalian cells in that neither the decrease in polysomes nor stress-granule formation required phosphorylation of eIF2α at the position homologous to that of serine 51 in mammalian eIF2α and in the occurrence of a novel XRNA-focus.

GENETIC MANIPULATION OF KINETOPLASTIDA

During the 1980s, many kinetoplastid genes were cloned and their function inferred from homology with genes from other organisms, location of the corresponding proteins or expression in heterologous systems. Up until 1990, before the availability of DNA transfection methodology, we could not analyze the function of kinetoplastid genes within the organisms themselves. Since then, it has become possible to create and complement mutants, to overexpress foreign proteins in the parasites, to knock out genes and even to switch off essential functions. However, these methods are not equally applicable in all parasites. Here, Christine Clayton highlights the differences and similarities between the most commonly used model organisms, and assesses the relative advantages of different approaches and parasites for different types of investigation.

Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p

Kinetoplastid protozoa confine large parts of glycolysis within glycosomes, which are microbodies related to peroxisomes. We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei glycosomes. The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains. It is targeted to peroxisomes when expressed in mammalian cells and yeast. The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Δ mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene. Sequence conservation is significant in the N‐ and C‐terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals. Several lines of evidence indicate that these domains are oriented towards the cytosol. TbPex11p can form homodimers, like its yeast counterpart. The TbPEX11 gene is essential in trypanosomes. Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm. Reduced expression results in trypanosomes with fewer, but larger, organelles.

Developmental regulation of a novel repetitive protein of Trypanosoma brucei.

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.

Metabolic compartmentation in African trypanosomes.

Differences between host and parasite energy metabolism are eagerly sought after as potential targets for antiparasite chemotherapy. In Kinetoplastia, the first seven steps of glycolysis are compartmented inside glycosomes, organelles that are related to the peroxisomes of higher eukaryotes. This arrangement is unique in the living world. In this review, Christine Clayton and Paul Michels discuss the implications of this unusual metabolic compartmentation for the regulation of trypanosome energy metabolism, and describe how an adequate supply of energy is maintained in different species and life cycle stages.