Biology subjects

Tiengwe, C.

Publications and source records attributed to Tiengwe, C..

7 recordsLinked to original sources

bioRxiv2026

DishCam: an open-source 3D-printed darkfield imager for microbial colonies and small organisms on Petri dishes

Petri dishes are routinely photographed using handheld cameras under reflected light. While adequate for large, distinct colonies, this approach fails to capture individual organisms or colonies that are transparent, do not reflect light well, or have fine structures, and is unsuitable for high quality timelapses. Darkfield illumination provides superior contrast for organisms on agar surfaces, but existing solutions rely on improvised setups with inconsistent lighting, or on large and expensive benchtop instruments that cannot be housed inside incubators. We present DishCam, an open-source device consisting entirely of 3D-printed parts and inexpensive off-the-shelf electronics, controlled by a Raspberry Pi running custom open-source software. DishCam produces high-contrast darkfield images using angled LED illumination beneath a Petri dish. All components used are off-the-shelf or easily 3D-printed. The camera and lights can be fully controlled through Raspberry Pi using our software. We demonstrate the device across multiple biological systems: Klebsiella aerogenes colonies of differing densities, Trypanosoma brucei colonies doing social motility, Dictyostelium discoideum streaming aggregation, and Caenorhabditis elegans on feeding substrates. The total cost of the basic device is approximately {pound}166 or $222, with the upgraded configuration costing approximately {pound}413 or $552, which includes a high-quality telephoto lens, SSD storage and incubator-compatible cables.

microbiology↗

bioRxiv2026

Chemotactic responses of Trypanosoma brucei procyclic forms to proline and other metabolites

Many protozoan parasites have complex life cycles entailing migration through different organs in their hosts, but the cues guiding them remain poorly understood. Using a semi-solid plate motility assay, we show that early procyclic forms of Trypanosoma brucei, the first stage to develop in the tsetse fly midgut, perceive several metabolites - including glucose, glycerol and proline - as chemoattractants, while the glycolytic end-product succinate acts as a repellent. During adaptation in the fly, T. brucei switches from glucose/glycerol to proline as its primary energy source. We show that the parasites chemotactic response towards proline requires adenylate cyclase ACP5 and the cyclic AMP response protein CARP3, two components of signalling pathway involved in pH sensing. These results further support a role for T. bruceis expanded repertoire of receptor adenylate cyclases as environmental sensors that guide navigation through the host. HighlightsO_LIThe plate assay reported here can be used to study how trypanosomes sense external signals and migrate in response to them. C_LIO_LIEarly procyclic forms of T. brucei respond to a variety of metabolites and exhibit different patterns of collective migration on plates, depending on the stimulus. C_LIO_LIThe response to proline operates through cAMP signalling and requires ACP5 and CARP3, two components also required for pH sensing. C_LI

microbiology↗

bioRxiv2026

A luminal proteome of the endoplasmic reticulum and Golgi apparatus reveals a novel modulator of ER stress tolerance in African trypanosomes

African trypanosomes employ specialised mechanisms of membrane trafficking as a key strategy to persist in both the mammalian host and insect vector. Their survival and pathogenicity rely on the continuous synthesis and surface delivery of extremely abundant surface coat proteins, imposing an extraordinary biosynthetic burden on the secretory pathway. Despite this, the luminal proteome of the T. brucei endoplasmic reticulum (ER) and Golgi apparatus remains incompletely characterised. Here, we exploit TurboID proximity biotinylation, using the abundant ER chaperone BiP (Binding-immunoglobulin protein) as luminal bait to map the ER proteome in bloodstream and procyclic lifecycle stages of Trypanosoma brucei. Comparison with BiPN, a truncated secretory form of BiP that transits the Golgi, provides differential compartmental labelling, together identifying 366 (BiP) and 428 (BiPN) proximity partners respectively and encompassing established ER quality control machinery, secretory cargo, and Golgi proteins. Quantitative ranking of BiP labelling intensity identifies a cohort of candidate BiP interactors: the most strongly enriched is Tb927.5.1160, a protein sharing structural homology with the mammalian BiP nucleotide exchange inhibitor MANF (mesencephalic astrocyte-derived neurotrophic factor). Endogenous mNeonGreen tagging confirms ER localisation of TbMANF in both life cycle stages, and reciprocal manipulation of its abundance by RNAi and inducible expression produces opposing shifts in cellular sensitivity to ER stress. These data are consistent with a role in regulating BiP ATPase cycling in an organism that, unlike yeast and mammals, lacks a canonical unfolded protein response, making TbMANF the first candidate regulator of BiP activity identified in kinetoplastids. Finally, TurboID proximity labelling anchored at the inner face of the nuclear pore via NUP65 extends our endomembrane map to the inner nuclear membrane, identifying candidate proteins of this specialised ER-continuous domain. AUTHOR SUMMARYAfrican sleeping sickness is caused by Trypanosoma brucei, a parasite that survives in the mammalian bloodstream by constantly renewing its protective protein coat. To synthesise and export this surface coat, the parasite relies on two intracellular compartments, the endoplasmic reticulum (ER) and the Golgi apparatus, which function as a quality control and sorting factory for proteins entering the secretory pathway. However, the identity of the proteins that populate these compartments in blood-stage parasites, and that maintain functioning under stress conditions, has remained poorly mapped. Here, we used an enzyme-based proximity labelling strategy that identifies neighbouring proteins in live cells without disturbing their targeting signals, generating a comprehensive protein inventory of both compartments across the two main T. brucei lifecycle stages. Among the most strongly labelled proteins was Tb927.5.1160, a protein structurally related to a mammalian regulator of the master ER chaperone BiP. Reducing or increasing the abundance of Tb927.5.1160 in parasites produced opposite changes in ER stress tolerance, identifying it as a candidate modulator of ER homeostasis in a lineage that regulates protein quality control through mechanisms distinct from those operating in yeast or human cells. Together, our findings provide a new molecular resource for understanding how T. brucei sustains secretory pathway function under the biosynthetic demands of mammalian and insect host infection.

biochemistry↗

bioRxiv2026

An octadecameric O-glucosyltransferase generates diversity in antibody epitopes on variant surface antigens in African trypanosomes

Immune evasion in many pathogens relies on sequence variation to generate antigenic diversity. African trypanosomes use an additional strategy where O-glucosylation of variant surface glycoproteins (VSGs) alters antibody epitopes and influences infection outcome, but the responsible enzyme remained elusive. Here, we show that an expression-site-associated gene (ESAG3), which is co-transcribed with the active VSG, encodes an O-glucosyltransferase required for VSG3 O-glycosylation in vivo. ESAG3 depletion abolishes VSG3 O-glycosylation and recognition of live trypanosomes by an O-glycan-dependent monoclonal antibody, while complementation restores both. ESAG3 has strict UDP-glucose donor specificity and modifies serine/threonine residues within VSG3 peptides in vitro. Cryo-electron microscopy at 3.4 Angstrom resolution reveals that ESAG3 forms a homo-octadecameric ring with C3 symmetry, an architecture not previously observed among glycosyltransferases. Structure-guided mutagenesis shows that disruption of ESAG3 inter-subunit interfaces reduces higher-order assembly, enhances substrate conversion, and alters VSG glycoform composition. Furthermore, we show that VSG3 O-glycan occupancy correlates with susceptibility to complement-mediated trypanolysis in an antibody-dependent manner, establishing that ESAG3-mediated O-glucosylation shapes both antibody specificity and the functional outcome of the host response.

microbiology↗

bioRxiv2024

A high-throughput protein tagging toolkit that retains endogenous UTRs for studying gene regulation in Kinetoplastids.

Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in Kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology. We present a CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from Thosea asigna to generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events. We validate the system by tagging six Trypanosoma brucei proteins and demonstrate: (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.

molecular biology↗

bioRxiv2024

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake; i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis. Author SummaryBlood-stage trypanosome parasites have a specialised invagination on the cell surface named the flagellar pocket (FP), where invariant essential nutrient receptors are located. The pocket houses diverse proteins, including a transferrin receptor (TfR), which facilitates uptake of host transferrin-bound iron for survival. Several FP proteins, including TfR, are linked to complex sugar molecules (carbohydrates), the functions of which are not well understood. Complex carbohydrates are made by enzymes called glycosyltransferases (GTs) and previously we partially inhibited complex carbohydrate synthesis by deletion of either TbGT8 or TbGT10. However, mutant parasites lacking either one of these enzymes survived, suggesting functional redundancy. Here, we created a parasite mutant that lacks both TbGT8 and TbG10 to understand the combined effect of losing both enzymes. The mutant parasites showed a decreased ability to uptake tomato lectin, a protein that specifically binds to these sugar conjugates in the FP, indicating a reduction in carbohydrate complexity. Despite reduced complexity in the sugar structures attached to TfR, its critical function in transferrin/iron uptake remained effective. Furthermore, the mutants remained viable in culture and in animal models, challenging previous assumptions about the necessity and function of these carbohydrate conjugates. Our findings imply a greater flexibility and redundancy in the carbohydrate complex roles than previously appreciated.

molecular biology↗

bioRxiv2021

Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation. Author SummaryAfrican trypanosomes are single-celled extracellular parasites of humans and animals relying on essential host nutrients for survival. They satisfy their iron needs by capturing host transferrin-bound iron using a surface-localised transferrin receptor (TfR) that is structurally distinct from its host counterpart. Little is known about the trypanosome response to fluctuations in host iron availability, with the exception of modulated TfR expression. We show that unlike other eukaryotes, at the transcriptome level, trypanosomes do not regulate iron-dependent enzymes as a mechanism to cope with iron deprivation. Instead, we identify a group of novel iron responsive trypanosome-specific genes, particularly an RNA Binding Protein RBP5 that is responsive to iron levels, albeit mediated by a distinct mechanism from TfR. We show that although RBP5 expression is elevated at the mRNA and protein levels, increased abundance above a maximum threshold is toxic. The trypanosome TfR has been suggested as a therapeutic target, but whether it is essential for optimal host colonisation is unclear. Our data demonstrate that trypanosomes efficiently import free iron from their environment independent of TfR suggesting that alternative iron uptake pathways exist, and that any therapeutic interventions targeting TfR must be evaluated with caution.

microbiology↗