A co-evolutionary arms race: trypanosomes shaping the human genome, humans shaping the trypanosome genome

<i>Trypanosoma brucei</i> is the causative agent of African sleeping sickness in humans and one of several pathogens that cause the related veterinary disease Nagana. A complex co-evolution has occurred between these parasites and primates that led to the emergence of trypanosome-specific defences and counter-measures. The first line of defence in humans and several other <i>catarrhine</i> primates is the trypanolytic protein apolipoprotein-L1 (APOL1) found within two serum protein complexes, trypanosome lytic factor 1 and 2 (TLF-1 and TLF-2). Two sub-species of <i>T. Brucei</i> have evolved specific mechanisms to overcome this innate resistance, <i>Trypanosoma brucei gambiense</i> and <i>Trypanosoma brucei rhodesiense</i>. In <i>T. b. Rhodesiense</i>, the presence of the serum resistance associated (SRA) gene, a truncated variable surface glycoprotein (VSG), is sufficient to confer resistance to lysis. The resistance mechanism of <i>T. b. Gambiense</i> is more complex, involving multiple components: reduction in binding affinity of a receptor for TLF, increased cysteine protease activity and the presence of the truncated VSG, <i>T. b. Gambiense</i>-specific glycoprotein <i>(TgsGP)</i>. In a striking example of co-evolution, evidence is emerging that primates are responding to challenge by <i>T. b. Gambiense</i> and <i>T. b. Rhodesiense</i>, with several populations of humans and primates displaying resistance to infection by these two sub-species

Control of endoderm development in Caenorhabditis elegans

The intestine (or gut) of Caenorhabditis elegans consists of 20 cells and is exclusively derived from the E-cell blastomere, by a precise pattern of cell divisions. During post- embryonic development no cell divisions occur in the E-lineage. Instead a nuclear division occurs, such that most cells are binucleate. In addition, endoreduplication occurs once during each larval stage, resulting in a DNA content of 32C in adult intestinal cell nuclei. Although much research has focussed on E-cell specification, not much is known about genes involved in the execution of the E-cell lineage. In this thesis, I have identified and characterised 4 classes of mutants with various E-cell lineage defects. These are mutants with an altered E-cell lineage, a mutant that fails to execute the LI nuclear division, mutants that undergo additional post embryonic nuclear divisions, and a mutant with an abnormal gut morphology. Most of the work in this thesis focussed on the class with an altered E-cell lineage. Two independent genes were identified in this class, Un-60(ij48) and lin-62(ij52), the behaviour of these mutant alleles is genetically very distinct, lin-60 is a maternal gene and ij48 is a gain-of-function allele whereas lin-62 is a zygotic gene and ij52 is a loss-of-function allele. By cell lineage analysis, I showed that extra intestinal cells were generated from the E blastomere in each mutant. Additionally, I have shown that the ij48 lesion creates deregulation of the cell cycle in the E-cell lineage, resulting in a hyperplasia of intestinal cells, while other aspects of intestinal cell function are retained. Using a novel RNA-mediated interference (RNAi) approach, I cloned the wild-type copy of the gene identified by lin-60(ij48) and found that it encoded a previously identified homologue of the cell cycle control gene, Cdc25 (cdc-25.1). While gain of cdc-25.1 created intestinal hyperplasia, loss of the wild-type cdc-25.1 by RNAi caused a failure of proliferation of intestinal and other cell types. I found that the site of the cdc-25.1 mutation in iJ48 mutants identifies a conserved motif However, the distribution of CDC-25.1 protein in ij48 animals does not explain the intestinal hyperplasia

Searching for novel cell cycle regulators in Trypanosoma brucei with an RNA interference screen

<b>Background</b><br /> The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference (RNAi) screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators.<p></p> <b>Results</b><br /> Procyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators.<p></p> <b>Conclusion</b><br /> Our study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.<p></p&gt

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter

Resolving the apparent transmission paradox of African sleeping sickness

Human African trypanosomiasis (HAT), or African sleeping sickness, is a fatal disease found throughout sub-Saharan Africa. The disease is close to elimination in many areas, although it was similarly close to elimination once before and subsequently reemerged, despite seemingly low rates of transmission. Determining how these foci persisted and overcame an apparent transmission paradox is key to finally eliminating HAT. By assessing clinical, laboratory, and mathematical data, we propose that asymptomatic infections contribute to transmission through the presence of an overlooked reservoir of skin-dwelling parasites. Our assessment suggests that a combination of asymptomatic and parasitaemic cases is sufficient to maintain transmission at foci without animal reservoirs, and we argue that the current policy not to treat asymptomatic HAT should be reconsidered

Fate specification and tissue-specific cell cycle control of the <i>Caenorhabditis elegans</i> intestine

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein β-transducin repeat-containing protein (β-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans β-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant

Macrophage migrating inhibitory factor expression is associated with Trypanosoma brucei gambiense infection and is controlled by trans-acting expression quantitative trait loci in the Guinean population

Infection by Trypanosoma brucei gambiense is characterized by a wide array of clinical outcomes, ranging from asymptomatic to acute disease and even spontaneous cure. In this study, we investigated the association between macrophage migrating inhibitory factor (MIF), an important pro-inflammatory cytokine that plays a central role in both innate and acquired immunity, and disease outcome during T. b. gambiense infection. A comparative expression analysis of patients, individuals with latent infection and controls found that MIF had significantly higher expression in patients (n=141; 1.25 +/- 0.07; p<.0001) and latent infections (n=25; 1.23 +/- 0.13; p=.0005) relative to controls (n=46; 0.94 +/- 0.11). Furthermore, expression decreased significantly after treatment (patients before treatment n=33; 1.40 +/- 0.18 versus patients after treatment n=33; 0.99 +/- 0.10, p=.0001). We conducted a genome wide eQTL analysis on 29 controls, 128 cases and 15 latently infected individuals for whom expression and genotype data were both available. Four loci, including one containing the chemokine CXCL13, were found to associate with MIF expression. Genes at these loci are candidate regulators of increased expression of MIF after infection. Our study is the first data demonstrating that MIF expression is elevated in T. b. gambiense-infected human hosts but does not appear to contribute to pathology

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporte

The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes

The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell divisio