Development of an improved transposon mutagenesis system for comprehensive genetic analysis of Borrelia burgdorferi

PROJECT SUMMARY/ABSTRACT Lyme disease, caused by Borrelia burgdorferi, remains a significant public health concern with limited treatment options. Despite decades of research, our knowledge of the genes it needs for transmission and infection is largely incomplete, hindering the development of effective therapies, vaccines, and diagnostic tests. In this proposal we aim to develop innovative high-throughput genetic tools that will facilitate the study of B. burgdorferi pathogenesis and accelerate Lyme disease research. We will create a high-efficiency transposon insertional mutagenesis system for B. burgdorferi, overcoming current limitations in generating comprehensive mutant libraries. This system will employ a custom-designed transposon delivery plasmid carrying a multifunctional Himar1 transposon. We will use transposon sequencing (Tn-seq) to conduct genome-wide fitness screens under various growth conditions, including different carbon sources relevant to the pathogen's life cycle. This will allow us to identify essential and conditionally essential genes for B. burgdorferi survival and growth. In addition, we use the transposon insertion library tool and Tn-seq to perform a comprehensive genetic interaction mapping of an important transcriptional regulator to begin to define its downstream regulated genes. By creating these genetic tools and resources, this project will advance the field of Lyme disease research. The methods developed here will enable rapid, systematic studies of B. burgdorferi gene function and regulation. The development of these tools will provide insights into B. burgdorferi's genetic regulatory networks and essential pathways to aid future research on targeted drug development for Lyme disease prevention and treatment.