The study of biomolecular condensates and other membraneless organelles has shifted the paradigm of cellular organisation, yet their role in parasitic biology remains an emerging field. While model eukaryotes provide the blueprint for phase separation and spatial compartmentalisation, kinetoplastid parasites offer a unique lens through which to explore the functional limits of these 'regulatory hubs'. In this opinion article, we argue that their unconventional biology renders them valuable models for understanding spatial regulation of gene expression. While Trypanosoma brucei serves as the current benchmark, significant gaps remain regarding condensate diversity across other kinetoplastids. We propose a broader investigation into whether these organisms utilise conserved condensates with divergent molecular compositions or entirely novel assemblies to drive their life cycle, pathogenesis, and immune evasion.

African trypanosomes are important parasites in sub-Saharan Africa that undergo a quorum-sensing dependent development to morphologically 'stumpy forms' in mammalian hosts to favour transmission by tsetse flies. However, some trypanosome clades have simplified their lifecycle by escaping dependence on tsetse allowing an expanded geographic range, with direct transmission between hosts achieved via blood-feeding biting flies and vampire bats (Trypanosoma brucei evansi, causing 'surra') or through sexual transmission (Trypanosoma brucei equiperdum, causing 'dourine'). Concomitantly, stumpy formation is reduced and the isolates are described as monomorphic, with infections spread widely in Africa, Asia, South America and parts of Europe. Here, using genomic analysis of distinct field isolates, we identify molecular changes that accompany the loss of the stumpy formation in monomorphic clades. Using CRISPR-mediated allelic replacement, mutations in two exemplar genes (Tb927.2.4020; Tb927.5.2580) are confirmed to reduce stumpy formation whereas another (Tb927.11.3400) is implicated in altered motility. Using laboratory selection we identify downregulation of RNA regulators as important in the initial development of monomorphism. This identifies a trajectory of events that simplify the life cycle in emergent and established monomorphic trypanosomes, with impact on disease spread, vector control strategies, geographical range and virulence.

Adaptation to a change of environment is an essential process for survival, in particular for parasitic organisms exposed to a wide range of hosts. Such adaptations include rapid control of gene expression through the formation of membraneless organelles composed of poly-A RNA and proteins. The African trypanosome Trypanosoma brucei is exquisitely sensitive to well-defined environmental stimuli that trigger cellular adaptations through differentiation events that characterise its complex life cycle. The parasite has been shown to form stress granules in vitro and it has been proposed that such a stress response could have been repurposed to enable differentiation and facilitate parasite transmission. Therefore, we explored the composition and positional dynamics of membraneless granules formed in response to starvation stress and during differentiation in the mammalian host between the replicative slender and transmission adapted stumpy forms. We find that T. brucei differentiation does not reflect the default response to environmental stress. Instead, the developmental response of the parasites involves a specific and programmed hierarchy of membraneless granule assembly, with distinct components and regulation by protein kinases such as TbDYRK, that are required for the parasite to successfully progress through its life cycle development and prepare for transmission.

Trypanosomatid parasitic protozoa are divergent from opisthokont models and have evolved unique mechanisms to regulate their complex life cycles and to adapt to a range of hosts. Understanding how these organisms respond, adapt, and persist in their different hosts could reveal optimal drug-control strategies. Protein kinases are fundamental to many biological processes such as cell cycle control, adaptation to stress, and cellular differentiation. Therefore, we have focused this review on the features and functions of protein kinases that distinguish trypanosomatid kinomes from other eukaryotes. We describe the latest research, highlighting similarities and differences between two groups of trypanosomatid parasites, Leishmania and African trypanosomes.

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo , we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro , procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals. Author summary African trypanosome parasites responsible for human and animal trypanosomiasis, live extracellularly in their mammalian host and exploit environmental information to regulate their virulence and transmissibility. Morphological slender forms that proliferate in the mammalian host transform into the non-proliferative stumpy forms by receiving an extracellular signal generated by the accumulating parasites. Parasites secrete peptidases into the host that degrade extracellular matrix (ECM) proteins. In this study, we evaluated the capacity to generate stumpy forms in vitro by including a basement membrane extract into the culture medium. We show that cells exposed to ECM proteins are able to differentiate efficiently into cell-cycle arrested stumpy forms and that the generated differentiation signal depends on the stumpy induction signalling pathway. This method can be used to generate stumpy forms in vitro , these being suitable for experimental analysis, thereby reducing the use of mice.

In the cell, reversible phosphorylation, controlled by protein phosphatases and protein kinases, initiates and regulates various signaling-dependent processes such as enzyme-substrate interactions, the cell cycle, differentiation, and immune responses. In addition to these processes, in unicellular parasites like Trypanosoma brucei, the causative agent of African sleeping sickness, additional signaling pathways have evolved to enable the survival of parasites in the changing environment of the vector and mammalian host. In this chapter, we describe two in vitro kinase assays and the use of the phosphoprotein chelator Phos-tag and show that these three polyacrylamide gel-based assays can be used for rapid target validation and detection of changes in phosphorylation.

Underlying dataset for the identifications of low-complexity regions (LCRs) in the proteome of Trypanosoma brucei. - Supplement File 2.xlsx (Position of every InterPro domain and LCR identified. All genes are provided with indication on chromosome localisation, presence of transmembrane domains, signal peptides and the localisation of the encoded proteins, either predicted using DeepLoc37 or observed (Tryptag38)). - Supplement File 3.xlsx (List of genes and Molecular Function gene ontology (GO) enrichment analysis of proteins with predicted LCRs in the N-terminal, central part or C-terminal or the different possible combinations.) - Supplement File 4.xlsx (Property analysis of sequences of every InterPro and LCRs identified.) - Supplement File 5.xlsx (List of genes and Molecular GO enrichment analysis of proteins presenting a Low (<8) or High (>9) polarity index level.) - Supplement File 6.xlsx (List and position of PTMs present on InterPro domains and LCRs. The different datasets from which the PTMs have been extracted can be found in the Zhang2020, Benz2019, Cayla2019, Urbaniak2013, Ooi2020, Fisk2012, Lott2012 and Moretti201712,13,15,17–19,27,28 columns. The sequence properties of the domains/LCRs on which these PTMs are located are also indicated. The list of modifications identified in Ooi et al. 202028 present on LCRs are indicated in the second sheet.) - Supplement File 7.xlsx (List and position of LCRs, signal peptides and their overlap.)

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 conformation 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.

Background: Low-complexity regions (LCRs) on proteins have attracted increasing attention recently due to their role in the assembly of membraneless organelles or granules by liquid-liquid phase separation. Several examples of such granules have been shown to sequester RNA and proteins in an inactive state, providing an important mechanism for dynamic post-transcriptional gene regulation. In trypanosome parasites, post-transcriptional control overwhelmingly dominates gene regulation due to the organisation of their genome into polycistronic transcription units. The purpose of the current study was to generate a substantially more comprehensive genome-wide survey of LCRs on trypanosome proteins than currently available . Methods: Using the Shannon's entropy method, provided in the R package 'entropy', we identified LCRs in the proteome of Trypanosoma brucei. Our analysis predicts LCRs and their positional enrichment in distinct protein cohorts and superimposes on this a range of post-translational modifications derived from available experimental datasets. Results: Our results highlight the enrichment of LCRs in the C-terminal region of predicted nucleic acid binding proteins, these acting as favoured sites for potential phosphorylation. Conclusions: The post-translational modifications of LCRs, and in particular the phosphorylation events, could contribute to post-transcriptional gene expression control and the dynamics of protein targeting to membraneless organelles in kinetoplastid parasites.

African trypanosomes cause human African trypanosomiasis and animal African trypanosomiasis. They are transmitted by tsetse flies in sub-Saharan Africa. Although most famous for their mechanisms of immune evasion by antigenic variation, there have been recent important studies that illuminate important aspects of the biology of these parasites both in their mammalian host and during passage through their tsetse fly vector. This Primer overviews current research themes focused on these parasites and discusses how these biological insights and the development of new technologies to interrogate gene function are being used in the search for new approaches to control the parasite. The new insights into the biology of trypanosomes in their host and vector highlight that we are in a 'golden age' of discovery for these fascinating parasites.

Trypanosoma brucei, the agents of African trypanosomiasis, undergo density-dependent differentiation in the mammalian bloodstream to prepare for transmission by tsetse flies. This involves the generation of cell-cycle arrested, quiescent, stumpy forms from proliferative slender forms. The signalling pathway responsible for the quorum sensing response has been catalogued using a genome-wide selective screen, providing a compendium of signalling protein kinases phosphatases, RNA binding proteins and hypothetical proteins. However, the ordering of these components is unknown. To piece together these components to provide a description of how stumpy formation arises we have used an extragenic suppression approach. This exploited a combinatorial gene knockout and overexpression strategy to assess whether the loss of developmental competence in null mutants of pathway components could be compensated by ectopic expression of other components. We have created null mutants for three genes in the stumpy induction factor signalling pathway (RBP7, YAK, MEKK1) and evaluated complementation by expression of RBP7, NEK17, PP1-6, or inducible gene silencing of the proposed differentiation inhibitor TbTOR4. This indicated that the signalling pathway is non-linear. Phosphoproteomic analysis focused on one pathway component, a putative MEKK, identified molecules with altered expression and phosphorylation profiles in MEKK1 null mutants, including another component in the pathway, NEK17. Our data provide a first molecular dissection of multiple components in a signal transduction cascade in trypanosomes.

Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association.

Leishmania parasites cause important human morbidity and mortality. Essential Leishmania genes escape genetic assessment by loss-of-function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the Herpes simplex virus thymidine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK4 as essential kinase in promastigotes. LmaMPK4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemotherapeutic intervention. A partial null mutant, expressing an MPK4 derivative with altered ATP-binding properties, showed defects in metacyclogenesis, establishing a first link of MPK4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in Leishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure-informed drug development.

Les protozoaires pathogenes du genre Leishmania ont developpe des mecanismes uniques de signalisation leur permettant de detecter des changements dans leur environnement. Ces changements vont permettre leur differenciation, etape essentielle pour assurer leur survie et leur croissance dans la cellule de l'hote mammifere. Peu de choses sont connues sur les mecanismes mis en jeu lors de la differenciation, mis a part le role essentiel joue par les MAP kinases (MAPKs) et la phosphorylation des proteines. Mes travaux de these ont permis i) de mettre en evidence les mecanismes de regulation de MPK10 chez Leishmania, qui sont tres inhabituels pour une MAPK, et ii) d'identifier un role potentiel de cette kinase dans la reponse a un appauvrissement du milieu en nutriments ou dans les etapes de differenciation des promastigotes en amastigotes axeniques. Les MAPKs sont activees par phosphorylation simultanee, par des MAPK kinases, des residus tyrosine et threonine du motif conserve TxY de la boucle d'activation, en reponse, par exemple, aux variations de conditions environnementales. En utilisant une approche transgenique j'ai observe i) une augmentation hautement regulee de la quantite et de l'activite de MPK10, durant la differenciation des promastigotes en amastigotes axeniques. En effet, l'activite de cette kinase doit etre inhibee apres les 48 premieres heures de differenciation pour permettre la survie et la proliferation des amastigotes axeniques ; ii) que l'activation de cette proteine n'etait pas correlee avec l'etat de phosphorylation de la tyrosine du motif TxY, qui est en grande partie constitutive malgre son role essentiel dans l'activite de la kinase. La regulation de l'activite de MPK10 semble dependre du domaine C-terminal. En effet, la suppression des derniers 46 acides amines de MPK10 entraine une augmentation de son activite, revelant ainsi le role du domaine C-terminal dans l'auto-inhibition de la kinase. J'ai egalement identifie une serine a la position 395 dans ce domaine C-terminal, dont la phosphorylation/dephosphorylation pourrait etre impliquee dans la regulation de MPK10. L'ensemble de ces resultats suggere que MPK10 est maintenue dans un etat partiellement ou pre- activee et auto-inhibee par son domaine C-terminal. Ce type de regulation est unique pour une MAPK et nous conduit a l'hypothese que MPK10 pourrait representer un interrupteur permettant une reponse rapide des parasites a des variations environnementales. En utilisant une approche complementaire basee sur la generation d'un mutant delete des alleles endogenes codant pour MPK10, j'ai montre que l'elimination de cette proteine ne semblait pas avoir de consequences sur la viabilite et la croissance des promastigotes en condition de culture normale. En revanche, l'absence de MPK10 semble avoir d'importantes consequences sur la viabilite des promastigotes lorsque ceux-ci sont cultives dans un milieu pauvre en nutriments. J'ai effectivement montre une correlation positive entre la quantite de MPK10 et la viabilite, car une diminution de la quantite de cette kinase entraine une augmentation de la mortalite cellulaire lors de la culture des promastigotes dans ce milieu pauvre. Toutes ces donnees indiquent que MPK10 presente un mode de regulation tres inhabituel et original compare a la regulation des autres MAPKs eucaryotes, tels qu'une conformation pre-activee intrinseque, et une regulation par auto-inhibition. De plus, MPK10 pourrait etre impliquee dans la detection des nutriments du milieu environnant.

Complement is a major innate immune defense against pathogens, tightly regulated to prevent host tissue damage. Atypical hemolytic uremic syndrome (aHUS) is characterized by endothelial damage leading to renal failure and is highly associated with abnormal alternative pathway regulation. We characterized the functional consequences of 2 aHUS-associated mutations (D(254)G and K(325)N) in factor B, a key participant in the alternative C3 convertase. Mutant proteins formed high-affinity C3-binding site, leading to a hyperfunctional C3 convertase, resistant to decay by factor H. This led to enhanced complement deposition on the surface of alternative pathway activator cells. In contrast to native factor B, the 2 mutants bound to inactivated C3 and induced formation of functional C3-convertase on iC3b-coated surface. We demonstrated for the first time that factor B mutations lead to enhanced C3-fragment deposition on quiescent and adherent human glomerular cells (GEnCs) and human umbilical vein endothelial cells (HUVECs), together with the formation of sC5b-9 complexes. These results could explain the occurrence of the disease, since excessive complement deposition on endothelial cells is a central event in the pathogenesis of aHUS. Therefore, risk factors for aHUS are not only mutations leading to loss of regulation, but also mutations, resulting in hyperactive C3 convertase.