Efficient and Stable Subcellular Protein Labeling in Leishmania mexicana Using a Re-Engineered mNeonGreen Integration
Tianyu Lei1, Mengtao Yu1, Panjing Lv1
1Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan 430030, China.
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The protozoan parasite Leishmania mexicana serves as a widely used model for studying trypanosomatid biology, yet the demand for stable, high-intensity fluorescent tools for precise subcellular protein localization remains unmet. In this study, we developed a versatile molecular toolbox by re-engineering the pLEXSY-hyg2.1 vector to express mNeonGreen (mNG), a next-generation fluorophore with superior brightness and photostability. Using a modular cloning strategy, we introduced a customized multiple cloning site (MCS) upstream of the mNG sequence to facilitate seamless N-terminal tagging of target proteins. The construct was integrated into the 18S rRNA locus via homologous recombination to ensure stable, constitutive expression. As a proof-of-concept, we fused a flagellar marker to the mNG reporter, resulting in a transgenic line exhibiting robust and specific subcellular fluorescence without compromising cellular fitness. Our results demonstrate that this integration-based system provides a highly efficient and stable platform for visualizing protein distribution within Leishmania. This tool significantly simplifies the generation of reporter strains and will facilitate high-resolution imaging studies of parasite organelle dynamics and functional genomics.
