PhD Scientific Days 2025

Poster Session II. - B: Molecular Medicine

Developing Next-Generation LTB4 Biosensors with a High-Throughput Molecular Pipeline

Name of the presenter

Vámosi Boldizsár

Institute/workplace of the presenter

Semmelweis University, Department of Phisiology

Authors

Boldizsár Vámosi¹, Benoit Roux¹, Klaudia Vágó-Kiss¹, Anna Török¹, Diána Kaszás¹, Fabian Dehne¹, Szimonetta Tamás¹, Balázs Enyedi¹

1: Semmelweis University, Department of Phisiology

Text of the abstract

Introduction: Leukotriene B4 (LTB4) is recognized for its crucial role as a chemoattractant during early stages of inflammation and wound healing. However, several questions about its production and spatiotemporal release from neutrophils remain unanswered. Previously, no tools were available for the real-time detection of LTB4 in live tissues. As a first step to overcome this challenge, we recently developed a genetically encoded fluorescent LTB4 biosensor. While GEM-LTB4 allows the basic visualization of LTB4 gradients both in vitro and in vivo, a second generation of sensors with wider dynamic range and greater sensitivity could significantly increase the versatility of the sensor. Additionally, a red-shifted variant would enable multiplexed imaging of parallel processes.
Aim: Our objective is to develop a novel LTB4 sensor designed for intravital microscopy using a high-throughput molecular biology pipeline.
Method: GEM-LTB4 was originally developed by the insertion of a circularly permuted green fluorescent protein flanked by few amino acid long linkers into BLT1, the endogenous receptor of LTB4. To develop a second generation of sensors, libraries encoding millions of sensor variants were generated by modifying the receptor, linker regions and the fluorescent protein. Furthermore, red sensor variants were also created by replacing cpEGFP with a conformationally sensitive RFP. These libraries were then screened on a single variant basis and the cells showing the highest fluorescence response triggered by LTB4 were isolated and sequenced.
Results: Previously, GEM-LTB4 demonstrated high sensitivity, detecting LTB4 concentrations as low as 10 nM. Upon stimulation with saturating LTB4 doses, GEM-LTB4 exhibited a maximal fluorescence increase of 103%. Following optimization using our new pipeline, we identified a red sensor variant with similar kinetics and a novel green sensor variant displaying a 3.5-fold greater fluorescence increase in response to the same LTB4 concentrations.
Conclusion: Using a novel development pipeline, we have generated a red-shifted and a novel green LTB4 sensor showing multiple-fold greater response to LTB4 compared to the original variant. Our optimization pipeline promises to be ideal for the development of novel fluorescent sensors for intravital microscopy.
Funding: EKÖP-2024-277 (Ministry for Culture and Innovation), HCEMM