Adaptation and Therapeutic Exploitation of the Plasma Membrane of African Trypanosomes

Quintana, Juan F.; Pino, Ricardo Canavate del; Yamada, Katsushi; Zhang, Ning; Field, Mark C.

doi:10.20944/preprints201806.0384.v1

Cited by 5 publications

(5 citation statements)

References 90 publications

(184 reference statements)

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“…These proteins were downregulated in the suramin-resistant isolate. These molecules actively degrade ISG75-suramin complex and subsequently release of suramin into the lysosome lumen ( Alsford et al, 2012 ; Quintana et al, 2018 ; de Koning, 2020 ). As such, the upregulation of ISG75 in the suramin resistant isolate is an indication that this parasite isolate can sufficiently take up suramin.…”

Section: Discussionmentioning

confidence: 99%

Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda

Bateta

Rono

Njunge

et al. 2021

International Journal for Parasitology: Drugs and Drug Resistan

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Human African Trypanosomiasis (HAT) is a disease of major economic importance in Sub-Saharan Africa. The HAT is caused by Trypanosoma brucei rhodesiense ( Tbr ) parasite in eastern and southern Africa, with suramin as drug of choice for treatment of early stage of the disease . Suramin treatment failures has been observed among HAT patients in Tbr foci in Uganda. In this study, we assessed Tbr parasite strains isolated from HAT patients responsive ( Tbr EATRO-232) and non-responsive ( Tbr EATRO-734) to suramin treatment in Busoga, Uganda for 1) putative role of suramin resistance in the treatment failure 2) correlation of suramin resistance with Tbr pathogenicity and 3) proteomic pathways underpinning the potential suramin resistance phenotype in vivo . We first assessed suramin response in each isolate by infecting male Swiss white mice followed by treatment using a series of suramin doses. We then assessed relative pathogenicity of the two Tbr isolates by assessing changes pathogenicity indices (prepatent period, survival and mortality). We finally isolated proteins from mice infected by the isolates, and assessed their proteomic profiles using mass spectrometry. We established putative resistance to 2.5 mg/kg suramin in the parasite Tbr EATRO-734. We established that Tbr EATRO-734 proliferated slower and has significantly enriched pathways associated with detoxification and metabolism of energy and drugs relative to Tbr EATRO-232. The Tbr EATRO-734 also has more abundantly expressed mitochondrion proteins and enzymes than Tbr EATRO-232. The suramin treatment failure may be linked to the relatively higher resistance to suramin in Tbr EATRO-734 than Tbr EATRO-232, among other host and parasite specific factors. However, the Tbr EATRO-734 appears to be less pathogenic than Tbr EATRO-232, as evidenced by its lower rate of parasitaemia. The Tbr EATRO-734 putatively surmount suramin challenges through induction of energy metabolism pathways. These cellular and molecular processes may be involved in suramin resistance in Tbr .

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Section: Discussionmentioning

confidence: 99%

Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda

Bateta

Rono

Njunge

et al. 2021

International Journal for Parasitology: Drugs and Drug Resistan

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“…The endocytic process and endosomal sorting regulatory mechanisms have been well studied in T. brucei [43,44]. Other studies, such as effects of silencing endocytosis regulators on cell morphology, may help to understand the possible mechanisms that link V-ATPase to the endocytic process.…”

Section: Discussionmentioning

confidence: 99%

Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei

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et al. 2020

Parasites Vectors

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Background: Vacuolar H +-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles. Beyond its role in pH maintenance, the involvement of V-ATPase in endocytosis is well documented in mammals and plants but is less clear in Trypanosoma brucei. Methods: In this study, the subcellular localization of V-ATPase subunit B (TbVAB) of T. brucei was assessed via in situ N-terminal YFP-tagging and immunofluorescence assays. Transgenic bloodstream forms (BSF) of T. brucei were generated which comprised either a V-ATPase subunit B (TbVAB) conditional knockout or a V-ATPase subunit A (TbVAA) knockdown. Acridine orange and BCECF-AM were employed to assess the roles of V-ATPase in the pH regulation of BSF T. brucei. The endocytic activities of three markers were also characterized by flow cytometry analyses. Furthermore, trypanosomes were counted from trypanolysis treatment groups (either containing 1% or 5% NHS) and endocytosed trypanosome lytic factor (TLF) was also analyzed by an immunoblotting assay. Results: TbVAB was found to localize to acidocalcisomes, lysosomes and probably also to endosomes of BSF of T. brucei and was demonstrated to be essential for cell growth. TbVAB depletion neutralized acidic organelles at 24 hours post-tetracycline depletion (hpd), meanwhile the steady state intracellular pH increased from 7.016 ± 0.013 to 7.422 ± 0.058. Trypanosomes with TbVAB depletion at 24 hpd were found to take up more transferrin (2.068 ± 0.277 fold) but less tomato lectin (49.31 ± 22.57%) by endocytosis, while no significant change was detected in dextran uptake. Similar endocytic dysregulated phenotypes were also observed in TbVAA knockdown cells. In addition, TbVAB depleted trypanosomes showed a low uptake of TLF and exhibited less sensitive to lysis in both 1% and 5% NHS treatments. Conclusions: TbVAB is a key component of V-ATPase and was found to play a key function in endocytosis as well as exhibiting different effects in a receptor/cargo dependent manner in BSF of T. brucei. Besides vacuolar alkalinization, the dysregulation of endocytosis in TbVAB depleted T. brucei is considered to contribute to the reduced sensitivity to lysis by normal human serum.

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“…Next, we sought evidence that TbAQP2 is ubiquitylated. Cycles of protein ubiquitylation and deubiquitylation are important for controlling the cell surface composition of trypanosomes [24,32,59]. Both proteasome-dependent and lysosome-mediated protein degradation are generally initiated by covalent attachment of one or more ubiquitin moieties to a substrate protein [60].…”

Section: Tbaqp2 Is Ubiquitylated and Degraded By The Lysosomementioning

confidence: 99%

“…There is also evidence that the three paralogs have differential locations within the parasite [12,20,22]. TbAQP2 and TbAQP3 are the product of a recent gene duplication and are unique to the African trypanosome lineage [12,20,22,24].…”

Section: Introductionmentioning

confidence: 99%

“…A selectivity filter restricts the size and properties of solutes that can effectively pass through the AQP pore [ 14 – 16 ]. In TbAQP1 and TbAQP3, this is formed by two constrictions of the channel: the canonical “NPA” within two half α-helices and a narrower “aromatic/arginine” (ar/R) motif ( Fig 1A ) [ 12 , 24 , 25 ]. Significantly, TbAQP2 does not retain this canonical configuration, but displays an unconventional “NPS/NSA” cation filter motif.…”

Section: Introductionmentioning

confidence: 99%

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Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei

et al. 2020

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Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/ glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmicoriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance.

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Adaptation and Therapeutic Exploitation of the Plasma Membrane of African Trypanosomes

Cited by 5 publications

References 90 publications

Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda

Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda

Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei

Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei

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