Localization And Functional Analysis On BBP1 Protein In Trypanosoma Brucei

Human African sleeping disease(HAT),one of the major tropical diseases monitored by WHO,is mainly reported in 36 countries in Africa.With the development of global trade and international tourism,three imported cases of HAT were also reported in China in 2015 and 2017.The pathogen of HAT,Trypanosoma brucei,is also a well-studied model organism in the field of pathology.T.brucei has numerous feathers distinct to mammalian cells,of which a pairwise structure called“basal bodies”(BBs),consisting of a matured basal body(mBB)and a pro-basal body(pBB),is structurally equivalent to a centrosome in mammals,and play important roles in cell division and flagella assembly.BBs are composed of hundreds of proteins,however,with many remaining unknown.Previous study in our laboratory has identified a basal body protein candidate,named BBP1,which is essential for cell growth,but lack of detailed information on its actual role.In this work,we performed detailed studies on its subcellular localization and managed to reveal its biological role in molecular level.Firstly,rabbit antiserum was generated with BBP1 peptide(Glu2-Glu400).The antibody titration for detecting BBP1 peptide was 1:5×10~6 and titration for detection for whole cell lysate was 1:10~5.The antiserum has been successfully applied in immunofluorescence.Antiserum immunofluorescence labeling and YFP in situ tagging showed that BBP1 signals appeared in mBB but not in the newly assembling pBB until at nuclear G2 phase.Secondly,BBP1 RNA interference cell line was generated and the cell growth was inhibited after 48 h RNAi,with 24.33%of cells showed abnormal DNA contents of one kinetoplast and multiple nucleus(1KXN).After 72 h for BBP1 RNAi,9.05%of cells were found with multiple kinetoplast and multiple nucleus(XKXN),together with the decrease of normal 1K1N/2K1N/2K2N cells(control,97.96%;24 h,92.43%;48 h,59.69%;72 h,34.22%).Appling DAPI staining,we were able to analyze the signal intensity of kinetoplast DNA,where inequal division of kinetoplast were found after 24h for BBP1 RNAi.Transmission electron microscopy(TEM)showed some cells lack of dimer microtubules or central singlet.Other phenotypes that assemble of SAS6 was impeded in the new pBB and the newly assembled flagellum failed to attach to cell body also appeared after BBP1 RNAi.The last,a hallmark characteristic of BBs is recruitment of tyrosinated α-tubulin,which could be checked by YL1/2 antibody labeling.As we found,the signal of YL1/2significant disappeared in mBBs after BBP1 RNAi,however,the localization and expression of recruitment associated protein,TbRP2,were not affected.In reverse,in TbRP2 RNAi cells,both the location of BBP1 and the recruitment of tyrosinated α-tubulin in BBs were affected.In summary,our results demonstrate that BBP1 is localized in both mBB and pBB,and is assembled into pBB during nuclear G2 phase.BBP1 plays an important role for pBB maturation and the growth of flagellum,and may further indirectly affects the division of kinetoplast and cytokinesis.Our study also suggests that BBP1 probably functions via affecting the recruitment of tyrosinated α-tubulin in BBs.