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Tag: Trypanosoma brucei

Mono-allelic epigenetic regulation of polycistronic transcription initiation by RNA polymerase II in Trypanosoma brucei

Donna Huber on December 20, 2024

TLF resistance correlates with HpHbR allelic expression

Unique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic transcription units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated and changes in gene expression are entirely post-transcriptional. Trypanosoma brucei brucei is unable to infect humans because of its susceptibility to an innate immune complex, trypanosome lytic factor (TLF) in the circulation of humans. The initial step in TLF-mediated lysis of T.b.brucei requires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here, we demonstrate that by in vitro selection with TLF, resistance is obtained in a stepwise process correlating with loss of HpHbR expression at an allelic level. RNA-seq, Pol II ChIP, and run-on analysis indicate HpHbR silencing is at the transcriptional level, where loss of Pol II binding at the promoter region specifically shuts down transcription of the HpHbR-containing gene cluster and the adjacent opposing gene cluster. Reversible transcriptional silencing of the divergent PTUs correlates with DNA base J modification of the shared promoter region. Base J function in establishing transcriptional silencing, rather than maintenance, is suggested by the maintenance of PTU silencing following the inhibition of J-biosynthesis and subsequent loss of the modified DNA base. Therefore, we show that epigenetic mechanisms exist to regulate gene expression via Pol II transcription initiation of gene clusters in a mono-allelic fashion. These findings suggest epigenetic chromatin-based regulation of gene expression is deeply conserved among eukaryotes, including early divergent eukaryotes that rely on polycistronic transcription.IMPORTANCEThe single-cell parasite Trypanosoma brucei causes lethal diseases in both humans and livestock. T. brucei undergoes multiple developmental changes to adapt in different environments during its digenetic life cycle. With protein-coding genes organized as polycistronic transcription and apparent absence of promoter-mediated regulation of transcription initiation, it is believed that developmental gene regulation in trypanosomes is essentially post-transcriptional. In this study, we found reversible Pol II transcriptional silencing of two adjacent polycistronic gene arrays that correlate with the novel DNA base J modification of the shared promoter region. Our findings support epigenetic regulation of Pol II transcription initiation as a viable mechanism of gene expression control in T. brucei. This has implications for our understanding how trypanosomes utilize polycistronic genome organization to regulate gene expression during its life cycle.

Rudo Kieft, Laura Cliffe, Haidong Yan, Robert J Schmitz, Stephen L Hajduk, Robert Sabatini. mBio. 2024 Dec 20:e0232824. doi: 10.1128/mbio.02328-24.

Acidocalcisome localization of membrane transporters and enzymes in Trypanosoma brucei

Donna Huber on October 9, 2024

FIG 1 Immunofluorescence microscopy of six membrane transporters.

 

Acidocalcisomes of Trypanosoma brucei are membrane-bounded organelles characterized by their acidity and high content of polyphosphate and cations, like calcium and magnesium. They have important roles in cation and phosphorus storage, osmoregulation, autophagy initiation, calcium signaling, and virulence. Acidocalcisomes of T. brucei possess several membrane transporters, pumps, and channels, some of which were identified by proteomic and immunofluorescence analyses and validated as acidocalcisome proteins by their colocalization with the acidocalcisome marker vacuolar proton pyrophosphatase (VP1). Here, we report that a set of membrane transporters and enzymes, which were proposed to be present in acidocalcisomes by the morphological appearance of tagged proteins, colocalize with VP1, validating their character as acidocalcisome proteins.

Importance: Acidocalcisomes are acidic organelles rich in polyphosphate and calcium present in a variety of eukaryotes and important for osmoregulation and calcium signaling. Several proteins were postulated to localize to acidocalcisomes based on their morphological characteristics. We provide validation of the localization of ten10 acidocalcisome proteins by their co-localization with enzymatic markers. These findings reveal the roles of acidocalcisomes in the storage of toxic metals, and the presence of enzymes involved in palmitoylation and polyphosphate metabolism.

Guozhong Huang, Roberto Docampo. Microbiol Spectr. 2024 Oct 9:e0112824. doi: 10.1128/spectrum.01128-24.

An X-Domain Phosphoinositide Phospholipase C (PI-PLC-like) of Trypanosoma brucei Has a Surface Localization and Is Essential for Proliferation

Donna Huber on February 28, 2023

Trypanosoma brucei is the causative agent of African trypanosomiasis, a deadly disease that affects humans and cattle. There are very few drugs to treat it, and there is evidence of mounting resistance, raising the need for new drug development. Here, we report the presence of a phosphoinositide phospholipase C (TbPI-PLC-like), containing an X and a PDZ domain, that is similar to the previously characterized TbPI-PLC1. TbPI-PLC-like only possesses the X catalytic domain and does not have the EF-hand, Y, and C2 domains, having instead a PDZ domain. Recombinant TbPI-PLC-like does not hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) and does not modulate TbPI-PLC1 activity in vitro. TbPI-PLC-like shows a plasma membrane and intracellular localization in permeabilized cells and a surface localization in non-permeabilized cells. Surprisingly, knockdown of TbPI-PLC-like expression by RNAi significantly affected proliferation of both procyclic and bloodstream trypomastigotes. This is in contrast with the lack of effect of downregulation of expression of TbPI-PLC1.

Núria W Negrão, Logan P Crowe, Brian S Mantilla, Rodrigo P Baptista, Sharon King-Keller, Guozhong Huang, Roberto Docampo. Pathogens. 2023 Feb 28;12(3):386. doi: 10.3390/pathogens12030386.

Chemical Optimization of CBL0137 for Human African Trypanosomiasis Lead Drug Discovery

Donna Huber on January 25, 2023

The carbazole CBL0137 (1) is a lead for drug development against human African trypanosomiasis (HAT), a disease caused by Trypanosoma brucei. To advance 1 as a candidate drug, we synthesized new analogs that were evaluated for the physicochemical properties, antitrypanosome potency, selectivity against human cells, metabolism in microsomes or hepatocytes, and efflux ratios. Structure-activity/property analyses of analogs revealed eight new compounds with higher or equivalent selectivity indices (5j, 5t, 5v, 5w, 5y, 8d, 13i, and 22e). Based on the overall compound profiles, compounds 5v and 5w were selected for assessment in a mouse model of HAT; while 5v demonstrated a lead-like profile for HAT drug development, 5w showed a lack of efficacy. Lessons from these studies will inform further optimization of carbazoles for HAT and other indications.

Baljinder Singh, Amrita Sharma, Naresh Gunaganti, Mitch Rivers, Pradip K Gadekar, Brady Greene, Michael Chichioco, Carlos E Sanz-Rodriguez, Courtney Fu, Catherine LeBlanc, Erin Burchfield, Nyle Sharif, Benjamin Hoffman, Gaurav Kumar, Andrei Purmal, Kojo Mensa-Wilmot, Michael P Pollastri. J Med Chem. 2023 Jan 25. doi: 10.1021/acs.jmedchem.2c01767.

Hypothesis-generating proteome perturbation to identify NEU-4438 and acoziborole modes of action in the African Trypanosome

Donna Huber on October 7, 2022

NEU-4438 is a lead for the development of drugs against Trypanosoma brucei, which causes human African trypanosomiasis. Optimized with phenotypic screening, targets of NEU-4438 are unknown. Herein, we present a cell perturbome workflow that compares NEU-4438’s molecular modes of action to those of SCYX-7158 (acoziborole). Following a 6 h perturbation of trypanosomes, NEU-4438 and acoziborole reduced steady-state amounts of 68 and 92 unique proteins, respectively. After analysis of proteomes, hypotheses formulated for modes of action were tested: Acoziborole and NEU-4438 have different modes of action. Whereas NEU-4438 prevented DNA biosynthesis and basal body maturation, acoziborole destabilized CPSF3 and other proteins, inhibited polypeptide translation, and reduced endocytosis of haptoglobin-hemoglobin. These data point to CPSF3-independent modes of action for acoziborole. In case of polypharmacology, the cell-perturbome workflow elucidates modes of action because it is target-agnostic. Finally, the workflow can be used in any cell that is amenable to proteomic and molecular biology experiments.

Amrita Sharma, Michael Cipriano, Lori Ferrins, Stephen L Hajduk, Kojo Mensa-Wilmot. iScience. 2022 Oct 7;25(11):105302. doi: 10.1016/j.isci.2022.105302. eCollection 2022 Nov 18.

Turnover of Variant Surface Glycoprotein in Trypanosoma brucei Is a Bimodal Process

Donna Huber on July 28, 2021

African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246-257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG+ extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [t1/2] of ∼26 h) but dropped significantly in the absence of GPI-PLC (t1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis. IMPORTANCE African trypanosomes, the protozoan agent of human African trypanosomaisis, avoid the host immune system by switching expression of the variant surface glycoprotein (VSG). VSG is a long-lived protein that has long been thought to be turned over by hydrolysis of its glycolipid membrane anchor. Recent work demonstrating the shedding of VSG-containing extracellular vesicles has led us to reinvestigate the mode of VSG turnover. We found that VSG is shed in part by glycolipid hydrolysis but also in approximately equal part by direct shedding of protein with intact lipid anchors. Shedding of exocytic vesicles made a very minor contribution to overall VSG turnover. These results indicate that VSG turnover is a bimodal process and significantly alter our understanding of the “life cycle” of this critical virulence factor.

Paige Garrison, Umaer Khan, Michael Cipriano, Peter J Bush, Jacquelyn McDonald, Aakash Sur, Peter J Myler, Terry K Smith, Stephen L Hajduk, James D Bangs. mBio. 2021 Jul 27;e0172521. doi: 10.1128/mBio.01725-21.

TbVps41 regulates trafficking of endocytic but not biosynthetic cargo to lysosomes of bloodstream forms of Trypanosoma brucei

Donna Huber on May 27, 2021

The bloodstream stage of Trypanosoma brucei, the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. Here we present evidence that RNAi-mediated expression down-regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket (“big eye” phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome-related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post-Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target.

Srinivasan Ramakrishnan, Rodrigo P Baptista, Beejan Asady, Guozhong Huang, Roberto Docampo. FASEB J. 2021 Jun;35(6):e21641. doi: 10.1096/fj.202100487R

Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome

Donna Huber on April 16, 2021

The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.

Catherine Sullenberger, Benjamin Hoffman, Justin Wiedeman, Gaurav Kumar, Kojo Mensa-Wilmot. PLoS One. 2021 Apr 16;16(4):e0249908. doi: 10.1371/journal.pone.0249908.

NUDIX hydrolases with inorganic polyphosphate exo- and endo-polyphosphatase activities in the glycosome, cytosol and nucleus of Trypanosoma brucei

Donna Huber on May 1, 2019

Trypanosoma brucei, a protist parasite that causes African trypanosomiasis or sleeping sickness, relies mainly on glycolysis for ATP production when in its mammalian host. Glycolysis occurs within a peroxisome-like organelle named the glycosome. Previous work from our laboratory reported the presence of significant amounts of inorganic polyphosphate (polyP), a polymer of three to hundreds of orthophosphate units, in the glycosomes and nucleoli of T. brucei In this work, we identified and characterized the activity of two Nudix hydrolases, TbNH2 and TbNH4, one located in the glycosomes and the other in the cytosol and nucleus, respectively, that can degrade polyP. We found that TbNH2 is an exopolyphosphatase with higher activity on short chain polyP, while TbNH4 is an endo- and exopolyphosphatase that has similar activity on polyP of various chain sizes. Both enzymes have higher activity at around pH 8.0. We also found that only TbNH2 can dephosphorylate ATP and ADP but with lower affinity than for polyP. Our results suggest that Nudix hydrolases can participate in polyP homeostasis and therefore may help control polyP levels in glycosomes, cytosol and nuclei of T. brucei.

Ciro D Cordeiro, Michael A Ahmed, Brian Windle, Roberto Docampo. 2019. Biosci Rep. 2019 May 1. pii: BSR20190894. doi: 10.1042/BSR20190894.