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Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface. Receptor expression is highest in developmental stages transmitted to the tsetse fly vector and those exposed to blood meals in the tsetse gut. Receptor gene deletion reduced tsetse infection, identifying this receptor as a virulence factor for transmission. This demonstrates how a pathogen evolved a molecular mechanism to increase transmission to an insect vector by exploitation of a mammalian complement regulator., African trypanosome infections can persist for years, but immune evasion mechanisms are not fully understood. Here, Macleod et al. identify a trypanosome receptor for mammalian factor H, a negative regulator of the alternative complement pathway, that increases parasite transmission to tsetse flies.

The sleeping sickness parasite,Trypanosoma brucei, uses quorum sensing (QS) to balance proliferation and transmission potential in the mammal bloodstream. A signal transduction cascade regulates this process, a component of which is a divergent member of the DYRK family of protein kinases, TbDYRK. Phylogenetic and mutational analysis in combination with activity and phenotypic assays revealed that TbDYRK exhibits a pre-activated confirmation and an atypical HxY activation loop motif, unlike DYRK kinases in other eukaryotes. Phosphoproteomic comparison of TbDYRK null mutants with wild type parasites identified molecules that operate on both the inhibitory ‘slender retainer’ and activatory ‘stumpy inducer’ arms of the QS control pathway. One of these molecules, the RNA-regulator TbZC3H20, regulates parasite QS, this being dependent on the integrity of its TbDYRK phosphorylation site. This analysis reveals fundamental differences to conventional DYRK family regulation and links trypanosome environmental sensing, signal transduction and developmental gene expression in a coherent pathway.

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics., Author summary Here we show that antibody-drug conjugates (ADCs) can be re-purposed from cancer immunotherapeutics to anti-protozoals by changing the specificity of the immunoglobulin to target a trypanosome cell surface receptor. Trypanosomes were used as a model system due to the availability of receptor null cell lines that allowed the unambiguous demonstration that ADCs targeted to a parasite surface receptor could be specifically internalised via receptor-mediated endocytosis. A single low dose of the resulting ADC was able to cure a stage 1 mouse model of trypanosome infection. We have used toxins and conjugation chemistry that are identical to anti-cancer ADCs demonstrating the ability to piggy-back onto the huge research efforts and resources that are being invested in the development of such ADCs. The potential for development of ADCs against a wide range of human pathogens is vast, where only epitope binding sites need vary in order to provide selectivity. This provides a far-reaching opportunity for the rapid development of novel anti-protozoals for the targeted killing of a wide range of pathogens that cause disease worldwide, especially in developing countries.

During their life cycle, trypanosomes must overcome conflicting demands to ensure their survival and transmission. First, they must evade immunity without overwhelming the host. Second, they must generate and maintain transmission stages at sufficient levels to allow passage into their tsetse vector. Finally, they must rapidly commit to onward development when they enter the tsetse fly. On the basis of recent quantification and modelling of Trypanosoma brucei infection dynamics, we propose that the interplay between immune evasion and development achieves both infection chronicity and transmissibility. Moreover, we suggest that a novel form of bistable regulation ensures developmental commitment on entry into the tsetse fly midgut.

Summary Sleeping sickness is characterized by waves of the extracellular parasite Trypanosoma brucei in host blood, with infections continuing for months or years until inevitable host death. These waves reflect the dynamic conflict between the outgrowth of a succession of parasite antigenic variants and their control by the host immune system. Although a contributor to these dynamics is the density-dependent differentiation from proliferative “slender forms” to transmissible “stumpy forms,” an absence of markers discriminating stumpy forms has prevented accurate parameterization of this component. Here, we exploit the stumpy-specific PAD1 marker, which functionally defines transmission competence, to quantitatively monitor stumpy formation during chronic infections. This allows reconstruction of the temporal events early in infection. Mathematical modeling of these data describes the parameters controlling trypanosome within-host dynamics and provides strong support for a quorum-sensing-like mechanism. Our data reveal the dominance of transmission stages throughout infection, a consequence being austere use of the parasite's antigen repertoire., Graphical Abstract Highlights ► Quantification of trypanosome transmission stages in chronic infections ► The temporal order of developmental events early in infection defined ► Mathematical data modeling provides support for parasite quorum sensing ► The dominance of transmission stages has key implications for VSG switch frequency

African trypanosomes produce different specialized stages for within-host replication and between-host transmission and therefore face a resource allocation trade-off between maintaining the current infection (survival) and investment into transmission (reproduction). Evolutionary theory predicts the resolution of this trade-off will significantly affect virulence and infectiousness. The application of life history theory to malaria parasites has provided novel insight into their strategies for survival and reproduction; how this framework can now be applied to trypanosomes is discussed. Specifically, predictions for how parasites trade-off investment in survival and transmission in response to variation in the within-host environment are outlined. An evolutionary approach has the power to explain why patterns of investment vary between strains and during infections, giving important insights into parasite biology.

A new mathematical model developed by Lythgoe et al. shows that the semi-predictable order of trypanosome antigenic variation can be generated by two parasite-intrinsic factors. The first is the different probabilities of antigen-gene activation that result from the different molecular mechanisms by which the genes become expressed. The second is the density-dependent differentiation of slender to stumpy cells. The study has important implications for understanding the dynamics of antigenic variation and for modelling the consequences of therapeutic strategies directed against trypanosomes.

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. DOI: http://dx.doi.org/10.7554/eLife.05553.001, eLife digest African Trypanosomes are a group of single-celled parasites that are a major concern for livestock farmers in sub-Saharan Africa. They are carried by the tsetse fly and can cause disease in domestic livestock that diminishes productivity through reduced growth, and may ultimately lead to death. The parasites are coated in a dense layer of protein that help them evade the host’s immune system by preventing immune cells from identifying them. Humans have evolved immunity to many trypanosome species by exploiting a weakness in their lifestyle. Trypanosomes need to get haem—a molecule found in the protein haemoglobin—from their host to survive. In blood plasma, haemoglobin is found associated with a carrier protein called haptoglobin. To acquire haem, the parasites have a protein called HpHbR that binds to these haptoglobin-haemoglobin ‘complexes’. However, in humans there are two complexes of proteins called TLFs that contain haemoglobin and a protein related to haptoglobin. The TLFs are also able to bind to HpHbR and are taken into the parasite. Once inside, TLFs cause internal compartments called lysosomes to swell, which leads to the death of the trypanosome. Two subspecies of Trypanosoma brucei are the only trypanosomes that infect humans as they can overcome the TLF1 defense. However, the details of how TLFs cause cell death at the molecular level are not clear. Lane-Serff et al. used a technique called x-ray crystallography to generate 3-D images of the HpHbR protein from T. brucei bound to the haptoglobin-haemoglobin complexes. These images show that HpHbR is elongated so that it only binds to haemoglobin and haptoglobin when they are together as a complex. The images also reveal that the shape of HpHbR enables it to hold apart the proteins in the protective layer that coats the trypanosome. This allows the haptoglobin-haemoglobin complex to bind to HpHbR, but in humans also makes HpHbR more likely to bind to TLF1. These findings may help to guide future efforts to protect humans and livestock from the diseases caused by trypanosomes. DOI: http://dx.doi.org/10.7554/eLife.05553.002

Here a genome-wide RNAi library screen is used to identify components of the signalling pathway that allow transformation of Trypanosoma brucei spp., the protozoan parasite responsible for important human and livestock diseases in sub-Saharan Africa, from proliferative slender forms to arrested stumpy forms which are transmitted to the tsetse fly vector. Trypanosoma brucei spp., the protozoan parasites responsible for human African trypanosomiasis and the livestock disease nagana in sub-Saharan Africa, proliferate as slender forms in the mammalian bloodstream then differentiate into to stumpy forms that are transmitted to the tsetse fly vector. This differentiation is regulated by a quorum-sensing-like process that depends on the 'stumpy induction factor' and a downstream signalling pathway, whose respective identity and competition have proven elusive. Here Keith Matthews and colleagues have used a genome-wide RNA interference library screen to identify the components of the signalling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified molecules could be possible targets for quorum-sensing interference, a strategy with antimicrobial potential against bacterial pathogens. The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub-Saharan Africa. In mammalian blood, two developmental forms of the parasite exist: proliferative ‘slender’ forms and arrested ‘stumpy’ forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission1. This response is triggered by an elusive ‘stumpy induction factor’ (SIF) whose intracellular signalling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently to SIF but can generate forms with stumpy characteristics when exposed to cell-permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNA interference library screen to identify the signalling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) or 8-pCPT-2′-O-methyl-5′-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide Ion Torrent based RNAi target sequencing identified cohorts of genes implicated in each step of the signalling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo. The putative RNA-binding protein, RBP7, was required for normal quorum sensing and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that quorum sensing signalling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for quorum-sensing interference-based therapeutics.

This work was supported by a Wellcome Trust Programme grant to KM and by a Wellcome Trust Strategic award to the Centre for Immunity, Infection and Evolution at the University of Edinburgh. SM was supported by a studentship from the Medical Research Council, UK. 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. Publisher PDF

Trypanosomatid parasites provide an extreme model for the posttranscriptional control of eukaryotic gene expression. However, most analysis of their differential gene regulation has focussed on comparisons between life-cycle stages that exist in the blood of mammalian hosts and tsetse flies, the parasite's vector. These environments differ acutely in their temperature, and nutritional, metabolic and molecular composition. In the bloodstream, however, a more exquisitely regulated developmental step occurs: the production of transmissible stumpy forms from proliferative slender forms. This transition occurs in the relatively homogenous bloodstream environment, with stumpy-specific gene expression being repressed until accumulation of a proposed parasite-derived signal, stumpy induction factor. Here, we have dissected the regulatory signals that repress the expression of the stumpy-specific surface transporter PAD1 in slender forms. Using transgenic parasites capable of stumpy formation we show that PAD1-repression is mediated by its 3'-untranslated region. Dissection of this region in monomorphic slender forms and pleomorphic slender and stumpy forms has revealed that two regulatory regions co-operate to repress PAD1 expression, this being alleviated on exposure to SIF in pleomorphs or cAMP analogues that act as stumpy induction factor mimics in monomorphs. These studies identify elements that regulate trypanosome gene expression during development in their mammalian host.

Trypanosomatid parasites are notorious for the human diseases they cause throughout Africa and South America. However, non-pathogenic trypanosomatids are also found worldwide, infecting a wide range of hosts. One example is Trypanosoma (Megatrypanum) theileri, a ubiquitous protozoan commensal of bovids, which is distributed globally. Exploiting knowledge of pathogenic trypanosomatids, we have developed Trypanosoma theileri as a novel vehicle to deliver vaccine antigens and other proteins to cattle. Conditions for the growth and transfection of T. theileri have been optimised and expressed heterologous proteins targeted for secretion or specific localisation at the cell interior or surface using trafficking signals from Trypanosoma brucei. In cattle, the engineered vehicle could establish in the context of a pre-existing natural T. theileri population, was maintained long-term and generated specific immune responses to an expressed Babesia antigen at protective levels. Building on several decades of basic research into trypanosomatid pathogens, Trypanosoma theileri offers significant potential to target multiple infections, including major cattle-borne zoonoses such as Escherichia coli, Salmonella spp., Brucella abortus and Mycobacterium spp. It also has the potential to deliver therapeutics to cattle, including the lytic factor that protects humans from cattle trypanosomiasis. This could alleviate poverty by protecting indigenous African cattle from African trypanosomiasis., Author Summary Single-celled parasites of the order Kinetoplastida are responsible for devastating diseases of humans and animals, including African trypanosomiasis, Chagas' disease and leishmaniasis. However, there are also many species of trypanosomatids that do not cause disease and are distributed globally. One example is Trypanosoma (Megatrypanum) theileri, which is restricted to bovids and ubiquitous in cattle herds worldwide. This organism is maintained extracellularly in the blood and tissues long-term without any observed ill effects on host health or productivity. Using knowledge of gene expression and protein trafficking in pathogenic trypanosomatids, we have successfully developed, from first principles, Trypanosoma theileri as a delivery system for vaccine antigens and therapeutics. Procedures for the growth, transfection and heterologous gene expression of T. theileri have been developed, and the delivery of a vaccine antigen derived from Babesia divergens evaluated in vivo. Our results demonstrate the ability of T. theileri to be used as a flexible and easily manipulated protein delivery system suitable for the control of cattle pathogens and cattle-borne zoonoses. In one notable application, we propose that the system could allow the expression of serum trypanolytic factors in cattle, with the potential to alleviate poverty in Africa through the killing of pathogenic trypanosomatids in livestock.

The sleeping sickness parasite, Trypanosoma brucei, must differentiate in response to the changing environments that it encounters during its complex life cycle. One developmental form, the bloodstream stumpy stage, plays an important role in infection dynamics and transmission of the parasite. Recent advances have shed light on the molecular mechanisms by which these stumpy forms differentiate as they are transmitted from the mammalian host to the insect vector of sleeping sickness, tsetse flies. These molecular advances now provide improved experimental tools for the study of stumpy formation and function within the mammalian bloodstream. They also offer new routes to therapy via high-throughput screens for agents that accelerate parasite development. Here, we shall discuss the recent advances that have been made and the prospects for future research now available.