Florimond Célia, Sahin Annelise, Vidilaseris Keni, Dong Gang, Landrein Nicolas, Dacheux Denis, Albisetti Anna, Byard Edward H, Bonhivers Mélanie, Robinson Derrick R
University Bordeaux, Microbiologie Fondamentale et Pathogenicité, Bordeaux, France; CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria.
PLoS Pathog. 2015 Mar 30;11(3):e1004654. doi: 10.1371/journal.ppat.1004654. eCollection 2015 Mar.
The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.
病原体布氏锥虫的鞭毛袋（FP）是一种重要的单拷贝结构，由表膜内陷形成。它是内吞和外排的独特位点，也是寄生虫致病性所必需的。鞭毛袋由不同的结构亚域组成，其中研究最少的是环状/马蹄形鞭毛袋环（FPC）。迄今为止，鞭毛袋环唯一已知的成分是蛋白质BILBO1，一种细胞骨架蛋白，其N端包含一个泛素样折叠、两个EF手结构域，以及一个大的C端卷曲螺旋结构域。已证明BILBO1能结合钙，但在本研究中我们证明，突变一个或两个钙结合结构域会阻止钙结合。在锥虫和哺乳动物细胞中表达缺失或突变形式的BILBO1表明，卷曲螺旋结构域对于BILBO1聚合物的形成是必要且充分的。酵母双杂交分析也支持这一点。在哺乳动物细胞中表达全长BILBO1会诱导形成具有逗号形和球形末端的线性聚合物，而经典钙结合结构域的突变会导致形成螺旋聚合物，两个EF手结构域的突变则会阻止线性聚合物的形成。我们还证明，在布氏锥虫中，卷曲螺旋结构域能够将BILBO1靶向鞭毛袋环并形成聚合物，而EF手结构域则影响聚合物的形状。这些数据表明，BILBO1具有内在的聚合物形成特性，结合钙可以调节这些聚合物的形式。我们讨论了这些特性是否会影响鞭毛袋环的形成。
