Search

In contrast to Trypanosoma brucei gambiense and T. b. rhodesiense (the causative agents of human African trypanosomiasis), T. b. brucei is lysed by apolipoprotein-L1 (apoL1)-containing human serum trypanolytic factors (TLF), rendering it non-infectious to humans. While the mechanisms of TLF1 uptake, apoL1 membrane integration, and T. b. gambiense and T. b. rhodesiense apoL1-resistance have been extensively characterised, our understanding of the range of factors that drive apoL1 action in T. b. brucei is limited. Selecting our bloodstream-form T. b. brucei RNAi library with recombinant apoL1 identified an array of factors that supports the trypanocidal action of apoL1, including six putative ubiquitin modifiers and several proteins putatively involved in membrane trafficking; we also identified the known apoL1 sensitivity determinants, TbKIFC1 and the V-ATPase. Most prominent amongst the novel apoL1 sensitivity determinants was a putative ubiquitin ligase. Intriguingly, while loss of this ubiquitin ligase reduces parasite sensitivity to apoL1, its loss enhances parasite sensitivity to TLF1-dominated normal human serum, indicating that free and TLF1-bound apoL1 have contrasting modes-of-action. Indeed, loss of the known human serum sensitivity determinants, p67 (lysosomal associated membrane protein) and the cathepsin-L regulator, ‘inhibitor of cysteine peptidase’, had no effect on sensitivity to free apoL1. Our findings highlight a complex network of proteins that influences apoL1 action, with implications for our understanding of the anti-trypanosomal action of human serum., Author summary Expression of the trypanolytic serum component, apolipoprotein-L1 (apoL1), by humans and the concomitant evolution of countermeasures by two African trypanosome sub-species defines their ability to cause disease in humans. A genome-scale RNAi screen identified more than 60 proteins that sensitise non-human infectious trypanosomes to apoL1. Comparing these outputs to previous screens that used different selection approaches provides insights into the mode-of-action of human serum-mediated killing of trypanosomes. The effectiveness of apoL1, and the trypanolytic factors (TLF) that carry it, is dependent on the parasite’s endocytic system for uptake, intracellular transit and delivery to target membranes. Comparing our current data to the outputs from previous screens revealed that apoL1 is able to exploit different routes to access its target membranes, depending on whether it is free or, as in human serum, complexed with TLF1 or TLF2.

Trypanosoma brucei, protozoan parasites that cause human African trypanosomiasis (HAT), depend on ornithine uptake and metabolism by ornithine decarboxylase (ODC) for survival. Indeed, ODC is the target of the WHO “essential medicine” eflornithine, which is antagonistic to another anti-HAT drug, suramin. Thus, ornithine uptake has important consequences in T. brucei, but the transporters have not been identified. We describe these amino acid transporters (AATs). In a heterologous expression system, TbAAT10-1 is selective for ornithine, whereas TbAAT2-4 transports both ornithine and histidine. These AATs are also necessary to maintain intracellular ornithine and polyamine levels in T. brucei, thereby decreasing sensitivity to eflornithine and increasing sensitivity to suramin. Consistent with competition for histidine, high extracellular concentrations of this amino acid phenocopied a TbAAT2-4 genetic defect. Our findings established TbAAT10-1 and TbAAT2-4 as the parasite ornithine transporters, one of which can be modulated by histidine, but both of which affect sensitivity to important anti-HAT drugs.—Macedo, J. P., Currier, R. B., Wirdnam, C., Horn, D., Alsford, S., Rentsch, D. Ornithine uptake and the modulation of drug sensitivity in Trypanosoma brucei.

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes., Author summary African trypanosomes are the causative agents of human sleeping sickness and Nagana cattle disease. These strictly extracellular pathogens multiply in the blood and body fluids of their mammalian hosts and the tsetse fly vector, where efficient redox regulation is essential for parasite survival. While most organisms use the glutathione/glutathione reductase and thioredoxin/thioredoxin reductase couples to maintain cellular redox balance, trypanosomes rely on a unique trypanothione-based thiol metabolism to survive exogenous and endogenous oxidative stresses. Despite the lack of thioredoxin reductases, the Trypanosoma brucei genome encodes thioredoxins, raising questions for their biological function. Our work is the first report on T. brucei thioredoxin-2 (Trx2). We show that Trx2 is located in the mitochondrion and its absence affects parasite proliferation and infectivity. Recombinant Trx2 lacks protein disulfide reductase activity but protects proteins against aggregation and maintains them folding-competent. Remarkably, a mutant that is devoid of any cysteine residues is able to fully substitute for the authentic protein under in vitro and in vivo conditions. Our data reveal that Trx2 does not function as a classical thioredoxin but acts as a chaperone that plays a crucial role in the mitochondrion of T. brucei.

Antivenom is an effective treatment of snakebite but, because of the complex interplay of fiscal, epidemiological, therapeutic efficacy and safety issues, the mortality of snakebite remains unacceptably high. Efficiently combating this high level of preventable death amongst the world's most disadvantaged communities requires the globally-coordinated action of multiple intervention programmes. This is the overall objective of the Global Snakebite Initiative. This paper describes the challenges facing the research community to develop snakebite treatments that are more efficacious, safe and affordable than current therapy.

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue- specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection. Refereed/Peer-reviewed

10 pages, 7 figures 1 table. Supporting Information: 2 figures, 2 tables., Venom is a critical evolutionary innovation enabling venomous snakes to become successful limbless predators; it is therefore vital that venomous snakes possess a highly efficient venom production and delivery system to maintain their predatory arsenal. Here, we exploit the unusual stability of messenger RNA in venom to conduct, for the first time, quantitative PCR to characterise the dynamics of gene expression of newly synthesised venom proteins following venom depletion. Quantitative PCR directly from venom enables real-time dynamic studies of gene expression in the same animals because it circumvents the conventional requirement to sacrifice snakes to extract mRNA from dissected venom glands. Using qPCR and proteomic analysis, we show that gene expression and protein re-synthesis triggered by venom expulsion peaks between days 3-7 of the cycle of venom replenishment, with different protein families expressed in parallel. We demonstrate that venom re-synthesis occurs very rapidly following depletion of venom stores, presumably to ensure venomous snakes retain their ability to efficiently predate and remain defended from predators. The stability of mRNA in venom is biologically fascinating, and could significantly empower venom research by expanding opportunities to produce transcriptomes from historical venom stocks and rare or endangered venomous species, for new therapeutic, diagnostic and evolutionary studies., This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship awarded to RB Currier and BBSRC research grant awarded to Dr. RA Harrison and Dr. SC Wagstaff (BB/F012675/1).

Bitis arietans is considered one of the most medically significant snakes in Africa, primarily due to a combination of its extensive geographical distribution, common occurrence and highly potent haemorrhagic and cytotoxic venom. Our investigation has revealed a remarkable degree of intra-species variation between pooled venom samples from different geographical origins across sub-Saharan Africa and Arabia, and within a group of individual specimens from the same origin in Nigeria as determined by a combination of immunological, biochemical and proteomic assays. We demonstrate significant quantitative and qualitative differences between B. arietans venom in terms of protein expression, immunogenicity and activity of snake venom metalloproteinases (SVMPs); toxins with a primary role in the haemorrhagic and tissue-necrotic pathologies suffered by envenomed victims. Specifically, we have identified a processed PII SVMP that exhibits striking inter-specimen variability.