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

Zhi-Shen Xu,Feng-Jun Li,De-Hua Lai,Geoff Hide,Zhao-Rong Lun

doi:10.1186/s13071-020-04068-4

Zhi-Shen Xu, Feng-Jun Li + Show 3 more

Open Access

https://doi.org/10.1186/s13071-020-04068-4

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Abstract

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 in Trypanosoma brucei.MethodsIn 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.ResultsTbVAB 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.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 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.

Highlights

Vacuolar H+-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles
Cell culture and transfection Trypanosoma brucei brucei cell lines Lister 427, SmOx B427 [24], SmOx PAntat1.1 and derivatives were grown in HMI-9 or SDM-79 media supplemented with 10% fetal calf serum (ExCell Bio, Shanghai, China)
the subcellular localization of V-ATPase subunit B (TbVAB) is localized in multiple organelles of bloodstream form trypanosomes and essential for cell growth To investigate the localization of vacuolar ATPase in the bloodstream forms (BSF) of T. brucei, a cell line expressing YFP-tagged vacuolar ATPase subunit B in situ was generated (TbVAB::YFP)

Summary

Introduction

Vacuolar H+-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles. Xu et al Parasites Vectors (2020) 13:214 efficient endocytic system that is capable of recycling the entire surface protein coat within 12 minutes [2]. It utilizes the serum-resistance associated (SRA) protein in T. b. V-ATPase is a conserved and crucial protein complex found in endosomal compartments in all eukaryotes [8, 9] This large complex contains many subunits, forming a rotary cytoplasmic V1 moiety (V1-ATPase, subunits A3B3CDE3FG3H) and a transmembrane Vo moiety (Vo proton channel, subunits ac8c′c′′de). The concerted action of the V 1 and Vo moiety couples ATP hydrolysis with the transport of protons across membranes and defines the fundamental function of V-ATPase in cellular compartment acidification. V-ATPase has been well studied in various model organisms, its roles and mechanisms of action in early-branched single cell protozoans is less clear

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