Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei
Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei
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液泡 ATP 酶耗竭导致布氏锥虫血流形式内吞作用失调
DOI：
10.1186/s13071-020-04068-4
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发表时间：
2020-04
影响因子：
3.2
通讯作者：
赖德华
中科院分区：
文献类型：
--
作者：
徐智深;Li Feng-Jun;Hide Geoff;伦照荣;赖德华
空泡H+-ATPase(V-ATPase)是一种高度保守的蛋白质复合体，它能水解三磷酸腺苷并泵出质子使空泡酸化。除了在pH维持中的作用外，V-ATPase在哺乳动物和植物中参与内吞作用的研究也很多，但在布鲁氏锥虫中还不清楚。采用N端YFP标记和免疫荧光法检测布鲁氏菌。转基因血流型(BSF)的研究很多。布鲁氏菌的产生包括V-ATPase亚单位B(TbVAB)条件性敲除或V-ATPase亚单位A(TbVAA)敲除。用吖啶橙和BCECF-AM研究V-ATPase在BSFT的pH调节中的作用。布鲁塞。用流式细胞仪分析三种标志物的内吞活性。用免疫印迹法检测内吞锥虫裂解因子(TLF)。结果TbVAB定位于BSF oft的酸性钙体、溶酶体，可能也定位于内含体。布鲁氏菌是细胞生长所必需的，TbVAB在四环素耗竭后24小时中和酸性细胞器，细胞内稳态pH从7.016±0.013升至7.422±0.058。TbVAB耗尽24 hpd的锥虫吞噬转铁蛋白(2.068±0.277倍)和减少番茄凝集素(49.31±22.57%)，而葡聚糖摄取无明显变化。在TbVA基因敲除细胞中也观察到类似的内吞异常表型。此外，在1%和5%的NHS处理中，TbVAB耗竭的锥体对TLF的摄取较低，对裂解的敏感性较低。结论TbVAB是V-ATPase的关键成分，在BSF oft中发挥着关键的内吞作用，并以受体/货物依赖的方式发挥不同的作用。布鲁塞。除空泡碱化外，TbVAB的内吞作用失调使T。布鲁氏菌被认为是导致正常人血清溶解敏感性降低的原因之一。
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.
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影响因子：
5.3
作者：
通讯作者：
--
影响因子：
3.6
作者：
Silverman JS;Schwartz KJ;Hajduk SL;Bangs JD
通讯作者：
Bangs JD
从细菌到人类细胞的细胞器保守的酸性蛋白质组学分析。
影响因子：
6.7
作者：
Huang G;Ulrich PN;Storey M;Johnson D;Tischer J;Tovar JA;Moreno SN;Orlando R;Docampo R
通讯作者：
Docampo R
锥形寄生虫中的钙信号传导。
影响因子：
4
作者：
Docampo R;Huang G
通讯作者：
Huang G
DOI：
10.1073/pnas.1505411112
发表时间：
2015-07-21
影响因子：
11.1
作者：
Baker, Nicola;Hamilton, Graham;Horn, David
通讯作者：
Horn, David