Etzensperger Ruth, Benninger Mattias, Pozzi Berta, Rehmann Ruth, Naguleswaran Arunasalam, Schumann Gabriela, Van Den Abbeele Jan, Roditi Isabel
Institute of Cell Biology, University of Bern, Bern, Switzerland.
Department of Biomedical Sciences, Trypanosoma Unit, Institute of Tropical Medicine, Antwerp, Belgium.
mBio. 2025 Feb 5;16(2):e0337524. doi: 10.1128/mbio.03375-24. Epub 2024 Dec 17.
Trypanosomes have different ways of communicating with each other. While communication via quorum sensing, or by the release and uptake of extracellular vesicles, is widespread in nature, the phenomenon of flagellar fusion has only been observed in . We showed previously that a small proportion of procyclic culture forms (corresponding to insect midgut forms) can fuse their flagella and exchange cytosolic and membrane proteins. This happens reproducibly in cell culture. It was not known, however, if flagellar fusion also occurs in the tsetse fly host, and at what stage of the life cycle. We have developed a split-Cre-Lox system to permanently label trypanosomes that undergo flagellar fusion. Specifically, we engineered trypanosomes to contain a GFP gene flanked by Lox sites in the reverse orientation to the promoter. In addition, the cells expressed inactive halves of the Cre recombinase, either N-terminal Cre residues 1-244 (N-Cre) or C-terminal Cre residues 245-343 (C-Cre). Upon flagellar fusion, these Cre halves were exchanged between trypanosomes, forming functional full Cre and flipping reverse-GFP into its forward orientation. We showed that cells that acquired the second half Cre through flagellar fusion were permanently modified and that the cells and their progeny constitutively expressed GFP. When tsetse flies were co-infected with N-Cre and C-Cre cells, GFP-positive trypanosomes were observed in the midgut and proventriculus 28-34 days post-infection. These results show that flagellar fusion not only happens in culture but also during the natural life cycle of trypanosomes in their tsetse fly host.
We have established a procedure to permanently label pairs of trypanosomes that transiently fuse their flagella and exchange proteins. When this occurs, a reporter gene is permanently flipped from the "off" to the "on" position, resulting in the production of green fluorescent protein. Crucially, green trypanosomes can be detected in tsetse flies co-infected with the two cell lines, proving that flagellar fusion occurs in the host. To our knowledge, we are the first to describe a split-Cre-Lox system for lineage tracing and selection in trypanosomes. In addition to its use in trypanosomes, this system could be adapted for other parasites and in other contexts. For example, it could be used to determine whether flagellar fusion occurs in related parasites such as Leishmania and or to monitor whether intracellular parasites and their hosts exchange proteins.
锥虫有不同的相互交流方式。虽然通过群体感应或通过细胞外囊泡的释放和摄取进行的交流在自然界中很普遍，但鞭毛融合现象仅在……中被观察到。我们之前表明，一小部分前循环培养形式（对应于昆虫中肠形式）可以融合它们的鞭毛并交换胞质和膜蛋白。这在细胞培养中可重复发生。然而，尚不清楚鞭毛融合是否也发生在采采蝇宿主中，以及在生命周期的哪个阶段发生。我们开发了一种分裂型Cre-Lox系统来永久标记经历鞭毛融合的锥虫。具体而言，我们对锥虫进行工程改造，使其含有一个GFP基因，该基因两侧是与启动子反向的Lox位点。此外，细胞表达Cre重组酶的无活性片段，即N端Cre残基1 - 244（N-Cre）或C端Cre残基245 - 343（C-Cre）。在鞭毛融合时，这些Cre片段在锥虫之间交换，形成有功能的完整Cre，并将反向GFP翻转到其正向。我们表明，通过鞭毛融合获得第二个Cre片段的细胞被永久修饰，并且这些细胞及其后代组成性表达GFP。当采采蝇同时感染N-Cre和C-Cre细胞时，在感染后28 - 34天的中肠和前胃中观察到GFP阳性锥虫。这些结果表明，鞭毛融合不仅发生在培养中，也发生在锥虫在采采蝇宿主的自然生命周期中。
我们建立了一种程序来永久标记短暂融合鞭毛并交换蛋白的锥虫对。当这种情况发生时，一个报告基因会从“关闭”永久翻转到“开启”位置，导致绿色荧光蛋白的产生。至关重要的是，在同时感染这两种细胞系的采采蝇中可以检测到绿色锥虫，证明鞭毛融合发生在宿主中。据我们所知，我们是第一个描述用于锥虫谱系追踪和筛选的分裂型Cre-Lox系统的人。除了用于锥虫外，该系统还可适用于其他寄生虫和其他情况。例如，它可用于确定相关寄生虫如利什曼原虫等是否发生鞭毛融合，或监测细胞内寄生虫与其宿主是否交换蛋白。
