Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei
| Autor: | Zhao-Rong Lun, Geoff Hide, De-Hua Lai, Zhi-Shen Xu, Feng-Jun Li |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Male
Vacuolar Proton-Translocating ATPases Endosome Protein subunit Intracellular pH Endocytic cycle Trypanosoma brucei brucei Protozoan Proteins Endosomes Biology Trypanosoma brucei Endocytosis lcsh:Infectious and parasitic diseases 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Animals Humans lcsh:RC109-216 030304 developmental biology chemistry.chemical_classification 0303 health sciences Trypanolysis Research Acridine orange Biological Transport Hydrogen-Ion Concentration Tetracycline biology.organism_classification Apolipoprotein L1 Recombinant Proteins Cell biology Infectious Diseases chemistry Transferrin Gene Knockdown Techniques Parasitology Female Rabbits Lysosomes Transcriptome 030217 neurology & neurosurgery Vacuolar ATPase |
| Zdroj: | Parasites & Vectors, Vol 13, Iss 1, Pp 1-12 (2020) Parasites & Vectors |
| ISSN: | 1756-3305 |
| DOI: | 10.1186/s13071-020-04068-4 |
| Popis: | BackgroundVacuolar 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 inTrypanosoma brucei.MethodsIn this study, the subcellular localization of V-ATPase subunit B (TbVAB) ofT. bruceiwas assessedvia in situN-terminal YFP-tagging and immunofluorescence assays. Transgenic bloodstream forms (BSF) ofT. bruceiwere 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 BSFT. 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.ResultsTbVAB was found to localize to acidocalcisomes, lysosomes and probably also to endosomes of BSF ofT. bruceiand was demonstrated to be essential for cell growth.TbVABdepletion 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 withTbVABdepletion 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 inTbVAAknockdown cells. In addition,TbVABdepleted trypanosomes showed a low uptake of TLF and exhibited less sensitive to lysis in both 1% and 5% NHS treatments.ConclusionsTbVAB 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 ofT. brucei. Besides vacuolar alkalinization, the dysregulation of endocytosis inTbVABdepletedT. bruceiis considered to contribute to the reduced sensitivity to lysis by normal human serum. |
| Databáze: | OpenAIRE |
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