Biology subjects

Wickstead, B.

Publications and source records attributed to Wickstead, B..

6 recordsLinked to original sources

bioRxiv2026

A platform for robust quantitative functional genomics reveals temporal fitness landscapes in African trypanosomes

Genome-scale phenotypic screening is transforming our understanding of host-pathogen interactions, but accurate quantitation of mutant fitness and generation of libraries in strains that capture complex disease characteristics remains challenging. Here we introduce Direct RNAi Fragment Sequencing (DRiF-Seq), a sequencing-optimized high-throughput RNA-interference platform that enables robust, clone-resolved quantification of fitness effects in African trypanosomes. Combined with an a posteriori noise estimation approach, DRiF-Seq provides reproducible estimates of fitness magnitude, timing, and statistical confidence for individual mutants and genes. Application to pleomorphic bloodstream-form Trypanosoma brucei enables the first genome-scale quantitative profiling in a mammalian infection model, creating a framework for investigating parasite biology at scale in host. DRiF-Seq demonstrates that over half of core genes have a fitness cost when targetted by RNAi, accurately resolves fitness landscapes within molecular complexes, identifies characteristic temporal signatures of gene disruption, and detects biologically meaningful differences between closely related parasite strains that predict differential drug sensitivity. Comparative analyses with genome-wide datasets from Toxoplasma and Plasmodium further demonstrate conserved and lineage-specific determinants of parasite fitness across eukaryotes. Together, DRiF-Seq provides a sensitive and transferable framework for quantitative functional genomics, a community resource for understanding parasite biology and a system for exploitation of high-complexity mutant screens in host.

Cell Biology↗

bioRxiv2023

A conserved trypanosomatid differentiation regulator controls substrate attachment and morphological development in Trypanosoma congolense

Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in that parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunodominant proteins, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission. Author summaryTrypanosoma congolense is a major cause of livestock trypanosomosis in sub-Saharan Africa where it contributes to lost economic productivity and poverty. These parasites differ in two key respects from the better-studied African trypanosomes, Trypanosoma brucei. Firstly, T. congolense adheres to surfaces in vitro and in the vaculature in vivo. Secondly, they lack a morphologically distinct transmission stage equivalent to the stumpy form of T. brucei. In the current work we identify a T congolense orthologue of a key developmental regulator in T. brucei, REG9.1, previously identified by a genome-wide RNAi screen. By RNA interference in transgenic T. congolense we demonstrate that TcREG9.1 functions both in parasite adherence in vitro and in vivo, and also in parasite development. The work provides a first insight into the unusual adherence and developmental biology of T. congolense and also suggests a possible model for how these characteristics interact to promote disease spread.

microbiology↗

bioRxiv2022

A synthetic ancestral Kinesin-13 depolymerises microtubules faster than any natural depolymerising kinesin

The activity of a kinesin is largely determined by the ~350 residue motor domain and this region alone is sufficient to classify a kinesin as a member of a particular family. The Kinesin-13 family are a group of microtubule depolymerising kinesins and are vital regulators of microtubule length. Members of this family are critical to spindle assembly and chromosome segregation in both mitotic and meiotic cell division and play crucial roles in cilium length control and neuronal development. To better understand the evolution of microtubule depolymerisation activity in the Kinesin-13 family, we created a synthetic ancestral Kinesin-13 motor domain. This phylogenetically-inferred ancestral motor domain is the sequence predicted to have existed in the common ancestor of the Kinesin-13 family. Here we show that the ancestral Kinesin-13 motor depolymerises stabilised microtubules faster than any previously tested depolymerase. This potent activity is more than an order of magnitude faster than the most highly studied Kinesin-13, MCAK and allows the ancestral Kinesin-13 to depolymerise doubly-stabilised microtubules that are unaffected by MCAK. These data suggest that the ancestor of the Kinesin-13 family was a super depolymeriser and that members of the Kinesin-13 family have evolved away from this extreme depolymerising activity to provide more controlled microtubule depolymerisation activity in extant cells.

molecular biology↗

bioRxiv2021

Variant surface glycoprotein expression in human African trypanosomiasis patients

Trypanosoma brucei gambiense is the primary causative agent of human African trypanosomiasis (HAT), a vector-borne disease endemic to West and Central Africa. The extracellular parasite evades antibody recognition within the host bloodstream by altering its Variant Surface Glycoprotein (VSG) coat through a process of antigenic variation. The serological tests which are widely used to screen for HAT use VSG as one of the target antigens. However, the VSGs expressed during human infection have not been characterized. Here we use VSG-seq to analyze the VSGs expressed in the blood of patients infected with T. b. gambiense and compared them to VSG expression in T. b. rhodesiense infections in humans as well as T. b. brucei infections in mice. The 44 VSGs expressed during T. b. gambiense infection revealed a striking bias towards expression of type B N-termini (82% of detected VSGs). This bias is specific to T. b. gambiense, which is unique among T. brucei subspecies in its chronic clinical presentation and anthroponotic nature, pointing towards a potential link between VSG expression and pathogenesis. The expressed T. b. gambiense VSGs also share very little similarity to sequences from 36 T. b. gambiense whole genome sequencing datasets, particularly in areas of the VSG protein exposed to host antibodies, suggesting that wild T. brucei VSG repertoires vary more than previously expected. Overall, this work demonstrates new features of antigenic variation in T. brucei gambiense and highlights the importance of understanding VSG repertoires in nature. Significance StatementHuman African Trypanosomiasis is a neglected tropical disease primarily caused by the extracellular parasite Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their Variant Surface Glycoprotein (VSG) coat. Despite the important role of VSGs in prolonging infection, VSG expression during human infections is poorly understood. A better understanding of natural VSG gene expression dynamics can clarify the mechanisms that T. brucei uses to alter its VSG coat and improve trypanosomiasis diagnosis in humans. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that there are features of antigenic variation unique to human-infective T. brucei subspecies and VSGs expressed in natural infection may vary more than previously expected.

microbiology↗

bioRxiv2021

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential drug sensitivity

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon drug sensitivity. Like T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly reduced in T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Comparative transcriptomics analysis showed higher levels of activity associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation and the succinate shunt in T. congolense. However, based on omics analysis and chemical inhibition, there does not appear to be significant levels of oxidative phosphorylation. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with pharmacological inhibitors, confirming a lack of essential electron transport chain activity in T. congolense, but increased sensitivity to inhibition of mitochondrial pyruvate import. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold greater EC50 against the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

microbiology↗

bioRxiv2019

Ndc80/Nuf2-like protein KKIP1 connects a stable kinetoplastid outer kinetochore complex to the inner kinetochore and responds to metaphase tension

Kinetochores perform an essential role in eukaryotes, coupling chromosomes to the mitotic spindle. In model organisms they are composed of a centromere-proximal inner kinetochore and an outer kinetochore network that binds to microtubules. In spite of its universal function, the composition of kinetochores in extant eukaryotes can differ greatly, and understanding how these different systems evolved and now function are important questions in cell biology. In trypanosomes and other Kinetoplastida, the kinetochores are extremely divergent, with most components showing no detectable similarity to proteins in other systems. They may also be very different functionally, potentially binding to the spindle directly via the inner kinetochore protein KKT4. However, we do not know the extent of the trypanosome kinetochore and proteins interacting with a highly divergent Ndc80/Nuf2-like protein, KKIP1, suggest the existence of more centromere-distal complexes. Here we use quantitative proteomics from multiple start-points to define a stable 9-protein kinetoplastid outer kinetochore (KOK) complex. Two of these core components were recruited from other nuclear processes, exemplifying the role of moonlighting proteins in kinetochore evolution. The complex is physically and biochemically distinct from KKT proteins, but KKIP1 links the inner and outer sets, with its C-terminus very close to the centromere and N-terminus at the outer kinetochore. Moreover, trypanosome kinetochores exhibit intra-kinetochore movement during metaphase, primarily by elongation of KKIP1, consistent with pulling at the outer kinetochores. Together, these data suggest that the KOK complex, KKIP5 and N-terminus of KKIP1 likely constitute the extent of the trypanosome outer kinetochore and that this assembly binds to the spindle with sufficient strength to stretch the kinetochore.

cell biology↗