Biology subjects

Carbajo, C. G.

Publications and source records attributed to Carbajo, C. G..

4 recordsLinked to original sources

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↗

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↗