How are Trypanosoma brucei receptors protected from host antibody-mediated attack?

Trypanosoma brucei is the causal agent of African Trypanosomiasis in humans and other animals. It maintains a long-term infection through an antigenic variation based population survival strategy. To proliferate in a mammal, T. brucei acquires iron and haem through the receptor mediated uptake of host transferrin and haptoglobin-hemoglobin respectively. The receptors are exposed to host antibodies but this does not lead to clearance of the infection. Here we discuss how the trypanosome avoids this fate in the context of recent findings on the structure and cell biology of the receptors.

Molecular mechanism of complement inhibition by the trypanosome receptor ISG65.

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.

Associative pyridinium electrolytes for air-tolerant redox flow batteries.

Pyridinium electrolytes are promising candidates for flow-battery-based energy storage1-4. However, the mechanisms underlying both their charge-discharge processes and overall cycling stability remain poorly understood. Here we probe the redox behaviour of pyridinium electrolytes under representative flow battery conditions, offering insights into air tolerance of batteries containing these electrolytes while providing a universal physico-chemical descriptor of their reversibility. Leveraging a synthetic library of extended bispyridinium compounds, we track their performance over a wide range of potentials and identify the singlet-triplet free energy gap as a descriptor that successfully predicts the onset of previously unidentified capacity fade mechanisms. Using coupled operando nuclear magnetic resonance and electron paramagnetic resonance spectroscopies5,6, we explain the redox behaviour of these electrolytes and determine the presence of two distinct regimes (narrow and wide energy gaps) of electrochemical performance. In both regimes, we tie capacity fade to the formation of free radical species, and further show that π-dimerization plays a decisive role in suppressing reactivity between these radicals and trace impurities such as dissolved oxygen. Our findings stand in direct contrast to prevailing views surrounding the role of π-dimers in redox flow batteries1,4,7-11 and enable us to efficiently mitigate capacity fade from oxygen even on prolonged (days) exposure to air. These insights pave the way to new electrolyte systems, in which reactivity of reduced species is controlled by their propensity for intra- and intermolecular pairing of free radicals, enabling operation in air.

Trypanosomes and complement: more than one way to die?

African trypanosomes show a remarkable ability to survive as extracellular parasites in the blood and tissue spaces of an infected mammal. Throughout the infection they are exposed to the molecules and cells of the immune system, including complement. In this opinion piece, we review decades-worth of evidence about how complement affects African trypanosomes. We highlight the discovery of a trypanosome receptor for complement C3 and we critically assess three recent studies which attempt to provide a structural and mechanistic view of how this receptor helps trypanosomes to survive in the presence of complement.

Shining the spotlight on the neglected: new high-quality genome assemblies as a gateway to understanding the evolution of Trypanosomatidae.

BACKGROUND: Protists of the family Trypanosomatidae (phylum Euglenozoa) have gained notoriety as parasites affecting humans, domestic animals, and agricultural plants. However, the true extent of the group's diversity spreads far beyond the medically and veterinary relevant species. We address several knowledge gaps in trypanosomatid research by undertaking sequencing, assembly, and analysis of genomes from previously overlooked representatives of this protistan group. RESULTS: We assembled genomes for twenty-one trypanosomatid species, with a primary focus on insect parasites and Trypanosoma spp. parasitizing non-human hosts. The assemblies exhibit sizes consistent with previously sequenced trypanosomatid genomes, ranging from approximately 18 Mb for Obscuromonas modryi to 35 Mb for Crithidia brevicula and Zelonia costaricensis. Despite being the smallest, the genome of O. modryi has the highest content of repetitive elements, contributing nearly half of its total size. Conversely, the highest proportion of unique DNA is found in the genomes of Wallacemonas spp., with repeats accounting for less than 8% of the assembly length. The majority of examined species exhibit varying degrees of aneuploidy, with trisomy being the most frequently observed condition after disomy. CONCLUSIONS: The genome of Obscuromonas modryi represents a very unusual, if not unique, example of evolution driven by two antidromous forces: i) increasing dependence on the host leading to genomic shrinkage and ii) expansion of repeats causing genome enlargement. The observed variation in somy within and between trypanosomatid genera suggests that these flagellates are largely predisposed to aneuploidy and, apparently, exploit it to gain a fitness advantage. High heterogeneity in the genome size, repeat content, and variation in chromosome copy numbers in the newly-sequenced species highlight the remarkable genome plasticity exhibited by trypanosomatid flagellates. These new genome assemblies are a robust foundation for future research on the genetic basis of life cycle changes and adaptation to different hosts in the family Trypanosomatidae.

Characterization of the major surface glycoconjugates of Trypanosoma theileri.

Trypanosoma theileri maintains a long-term extracellular infection with a low parasitaemia in bovids. The surface of this parasite is predicted to be decorated with several surface molecules including membrane surface proteases (MSPs), trans-sialidases and T. theileri putative surface proteins (TTPSPs). However, there are no experimental data to verify this hypothesis. Here, we have purified and partially characterized the surface glycoconjugates of T. theileri using biochemical and mass spectrometry-based approaches. The glycoconjugates fall into two classes: glycoproteins and glycolipids. Proteomic analysis of the glycoprotein fraction demonstrated the presence of MSPs and abundant mucin-like TTPSPs, with most predicted to be GPI-anchored. Mass spectrometric characterization of the glycolipid fraction showed that these are mannose- and galactose-containing glycoinositolphospholipids (GIPLs) that are larger and more diverse than those of its phylogenetic relative T. cruzi, containing up to 10 hexose residues and carrying either alkylacyl-phosphatidylinositol or inositol-phospho-ceramide (IPC) lipid components.

Decoding the impact of nuclear organization on antigenic variation in parasites.

Antigenic variation as a strategy to evade the host adaptive immune response has evolved in divergent pathogens. Antigenic variation involves restricted, and often mutually exclusive, expression of dominant antigens and a periodic switch in antigen expression during infection. In eukaryotes, nuclear compartmentalization, including three-dimensional folding of the genome and physical separation of proteins in compartments or condensates, regulates mutually exclusive gene expression and chromosomal translocations. In this Review, we discuss the impact of nuclear organization on antigenic variation in the protozoan pathogens Trypanosoma brucei and Plasmodium falciparum. In particular, we highlight the relevance of nuclear organization in both mutually exclusive antigen expression and genome stability, which underlie antigenic variation.

A unique mRNA decapping complex in trypanosomes.

Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.

Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei.

Trypanosoma brucei is a model trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is challenging because, as with most eukaryotic microbes, ~50% of genome-encoded proteins have completely unknown functions. Here, using fluorescence microscopy and cell lines expressing endogenously tagged proteins, we mapped the subcellular localization of 89% of the T. brucei proteome, a resource we call TrypTag. We provide clues to function and define lineage-specific organelle adaptations for parasitism, mapping the ultraconserved cellular architecture of eukaryotes, including the first comprehensive 'cartographic' analysis of the eukaryotic flagellum, which is vital for morphogenesis and pathology. To demonstrate the power of this resource, we identify novel organelle subdomains and changes in molecular composition through the cell cycle. TrypTag is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.

Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei.

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.

Invariant surface glycoprotein 65 of Trypanosoma brucei is a complement C3 receptor.

African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.

Black-necked spitting cobra (Naja nigricollis) phospholipases A2 may cause Trypanosoma brucei death by blocking endocytosis through the flagellar pocket.

African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host's adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components, including proteases and phospholipases A2. Here, we investigated the effect of Naja nigricollis venom on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VSG hydrolysis, however, N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) cell cycle arrest prior to cytokinesis. Our results indicate that a single protein family, the phospholipases A2 present in N. nigricollis venom, may be necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.

A longitudinal two-year survey of the prevalence of trypanosomes in domestic cattle in Ghana by massively parallel sequencing of barcoded amplicons.

BACKGROUND: Animal African Trypanosomiasis (AAT) is one of the most economically important diseases affecting livestock productivity in sub-Saharan Africa. The disease is caused by a broad range of Trypanosoma spp., infecting both wild and domesticated animals through cyclical and mechanical transmission. This study aimed to characterize trypanosomes present in cattle at regular intervals over two years in an AAT endemic and a non-endemic region of Ghana. METHODOLOGY/PRINCIPAL FINDINGS: Groups of cattle at Accra and Adidome were selected based on their geographical location, tsetse fly density, prevalence of trypanosomiasis and the breed of cattle available. Blood for DNA extraction was collected at approximately four to five-week intervals over a two-year period. Trypanosome DNA were detected by a sensitive nested PCR targeting the tubulin gene array and massively parallel sequencing of barcoded amplicons. Analysis of the data was a semi-quantitative estimation of infection levels using read counts obtained from the sequencing as a proxy for infection levels. Majority of the cattle were infected with multiple species most of the time [190/259 (73%) at Adidome and 191/324 (59%) at Accra], with T. vivax being the most abundant. The level of infection and in particular T. vivax, was higher in Adidome, the location with a high density of tsetse flies. The infection level varied over the time course, the timings of this variation were not consistent and in Adidome it appeared to be independent of prophylactic treatment for trypanosome infection. Effect of gender or breed on infection levels was insignificant. CONCLUSIONS/SIGNIFICANCE: Most cattle were infected with low levels of several trypanosome species at both study sites, with T. vivax being the most abundant. The measurements of infection over time provided insight to the importance of the approach in identifying cattle that could suppress trypanosome infection over an extended time and may serve as reservoir.

Identification of sequence-specific promoters driving polycistronic transcription initiation by RNA polymerase II in trypanosomes.

Protein-coding genes in trypanosomes occur in polycistronic transcription units (PTUs). How RNA polymerase II (Pol II) initiates transcription of PTUs has not been resolved; the current model favors chromatin modifications inducing transcription rather than sequence-specific promoters. Here, we uncover core promoters by functional characterization of Pol II peaks identified by chromatin immunoprecipitation sequencing (ChIP-seq). Two distinct promoters are located between divergent PTUs, each driving unidirectional transcription. Detailed analysis identifies a 75-bp promoter that is necessary and sufficient to drive full reporter expression and contains functional motifs. Analysis of further promoters suggests transcription initiation is regulated and promoters are either focused or dispersed. In contrast to the previous model of unregulated and promoter-independent transcription initiation, we find that sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes PTUs. These findings in Trypanosoma brucei suggest that in addition of chromatin modifications, promoter motifs-based regulation of gene expression is deeply conserved among eukaryotes.

Molecular detection of novel Anaplasma sp . and zoonotic hemopathogens in livestock and their hematophagous biting keds (genus Hippobosca) from Laisamis, northern Kenya.

Background: Livestock are key sources of livelihood among pastoral communities. Livestock productivity is chiefly constrained by pests and diseases. Due to inadequate disease surveillance in northern Kenya, little is known about pathogens circulating within livestock and the role of livestock-associated biting keds (genus Hippobosca) in disease transmission. We aimed to identify the prevalence of selected hemopathogens in livestock and their associated blood-feeding keds. Methods: We randomly collected 389 blood samples from goats (245), sheep (108), and donkeys (36), as well as 235 keds from both goats and sheep (116), donkeys (11), and dogs (108) in Laisamis, Marsabit County, northern Kenya. We screened all samples for selected hemopathogens by high-resolution melting (HRM) analysis and sequencing of PCR products amplified using primers specific to the genera: Anaplasma, Trypanosoma, Clostridium, Ehrlichia, Brucella, Theileria, and Babesia. Results: In goats, we detected Anaplasma ovis (84.5%), a novel Anaplasma sp. (11.8%), Trypanosoma vivax (7.3%), Ehrlichia canis (66.1%), and Theileria ovis (0.8%). We also detected A. ovis (93.5%), E. canis (22.2%), and T. ovis (38.9%) in sheep. In donkeys, we detected ' Candidatus Anaplasma camelii' (11.1%), T. vivax (22.2%), E. canis (25%), and Theileria equi (13.9%). In addition, keds carried the following pathogens; goat/sheep keds - T. vivax (29.3%) , Trypanosoma evansi (0.86%), Trypanosoma godfreyi (0.86%), and E. canis (51.7%); donkey keds - T. vivax (18.2%) and E. canis (63.6%); and dog keds - T. vivax (15.7%), T. evansi (0.9%), Trypanosoma simiae (0.9%) , E. canis (76%), Clostridium perfringens (46.3%), Bartonella schoenbuchensis (76%), and Brucella abortus (5.6%). Conclusions: We found that livestock and their associated ectoparasitic biting keds carry a number of infectious hemopathogens, including the zoonotic B. abortus. Dog keds harbored the most pathogens, suggesting dogs, which closely interact with livestock and humans, as key reservoirs of diseases in Laisamis. These findings can guide policy makers in disease control.

Transmission of 'Candidatus Anaplasma camelii' to mice and rabbits by camel-specific keds, Hippobosca camelina.

Anaplasmosis, caused by infection with bacteria of the genus Anaplasma, is an important veterinary and zoonotic disease. Transmission by ticks has been characterized but little is known about non-tick vectors of livestock anaplasmosis. This study investigated the presence of Anaplasma spp. in camels in northern Kenya and whether the hematophagous camel ked, Hippobosca camelina, acts as a vector. Camels (n = 976) and > 10,000 keds were sampled over a three-year study period and the presence of Anaplasma species was determined by PCR-based assays targeting the Anaplasmataceae 16S rRNA gene. Camels were infected by a single species of Anaplasma, 'Candidatus Anaplasma camelii', with infection rates ranging from 63-78% during the dry (September 2017), wet (June-July 2018), and late wet seasons (July-August 2019). 10-29% of camel keds harbored 'Ca. Anaplasma camelii' acquired from infected camels during blood feeding. We determined that Anaplasma-positive camel keds could transmit 'Ca. Anaplasma camelii' to mice and rabbits via blood-feeding. We show competence in pathogen transmission and subsequent infection in mice and rabbits by microscopic observation in blood smears and by PCR. Transmission of 'Ca. Anaplasma camelii' to mice (8-47%) and rabbits (25%) occurred readily after ked bites. Hence, we demonstrate, for the first time, the potential of H. camelina as a vector of anaplasmosis. This key finding provides the rationale for establishing ked control programmes for improvement of livestock and human health.

Sequential production of gametes during meiosis in trypanosomes.

Meiosis is a core feature of eukaryotes that occurs in all major groups, including the early diverging excavates. In this group, meiosis and production of haploid gametes have been described in the pathogenic protist, Trypanosoma brucei, and mating occurs in the salivary glands of the insect vector, the tsetse fly. Here, we searched for intermediate meiotic stages among trypanosomes from tsetse salivary glands. Many different cell types were recovered, including trypanosomes in Meiosis I and gametes. Significantly, we found trypanosomes containing three nuclei with a 1:2:1 ratio of DNA contents. Some of these cells were undergoing cytokinesis, yielding a mononucleate gamete and a binucleate cell with a nuclear DNA content ratio of 1:2. This cell subsequently produced three more gametes in two further rounds of division. Expression of the cell fusion protein HAP2 (GCS1) was not confined to gametes, but also extended to meiotic intermediates. We propose a model whereby the two nuclei resulting from Meiosis I undergo asynchronous Meiosis II divisions with sequential production of haploid gametes.

Diversity-oriented synthesis of polymer membranes with ion solvation cages.

Microporous polymers feature shape-persistent free volume elements (FVEs), which are permeated by small molecules and ions when used as membranes for chemical separations, water purification, fuel cells and batteries1-3. Identifying FVEs that have analyte specificity remains a challenge, owing to difficulties in generating polymers with sufficient diversity to enable screening of their properties. Here we describe a diversity-oriented synthetic strategy for microporous polymer membranes to identify candidates featuring FVEs that serve as solvation cages for lithium ions (Li+). This strategy includes diversification of bis(catechol) monomers by Mannich reactions to introduce Li+-coordinating functionality within FVEs, topology-enforcing polymerizations for networking FVEs into different pore architectures, and several on-polymer reactions for diversifying pore geometries and dielectric properties. The most promising candidate membranes featuring ion solvation cages exhibited both higher ionic conductivity and higher cation transference number than control membranes, in which FVEs were aspecific, indicating that conventional bounds for membrane permeability and selectivity for ion transport can be overcome4. These advantages are associated with enhanced Li+ partitioning from the electrolyte when cages are present, higher diffusion barriers for anions within pores, and network-enforced restrictions on Li+ coordination number compared to the bulk electrolyte, which reduces the effective mass of the working ion. Such membranes show promise as anode-stabilizing interlayers in high-voltage lithium metal batteries.

VSG mRNA levels are regulated by the production of functional VSG protein.

The bloodstream form of Trypanosoma brucei persists in mammalian hosts through a population survival strategy depending on antigenic variation of a cell surface coat composed of the variant surface glycoprotein (VSG). The integrity of the VSG coat is essential and blocking its synthesis results in a cell division cycle arrest just prior to cytokinesis. This observation indicates that VSG levels are monitored and that the cell has mechanisms to respond to a disruption of synthesis. Here, the regulation of VSG mRNA levels has been investigated by first measuring VSG mRNA copy number, and second using ectopic expression of VSG transgenes containing premature termination codons. The findings are that (i) VSG mRNA copy number varies with the identity of the VSG and (ii) a pathway detects synthesis of non-functional VSG protein and results in an increase in VSG mRNA levels.

Erratum to "The cell surface - A new journal for transkingdom cell wall research" [Cell Surface 1 (2018) 1].

[This corrects the article DOI: 10.1016/j.tcsw.2017.05.001.].