Abstract
Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe inflammatory pneumonia in humans called Legionnaires' disease, which results from bacterial replication within alveolar macrophages. L. pneumophila replication within macrophages is dependent on hundreds of individual protein virulence factors. Understanding how these virulence factors contribute to disease in an animal model is important to reveal aspects of host-pathogen interactions. High-throughput sequencing (HTS)-based screens using transposon (Tn) mutagenesis are powerful approaches to identify bacterial genes important for host-pathogen interactions. Since large libraries of Tn mutants are at risk of bottleneck effects, phenotypic screening of smaller numbers of targeted mutants is an effective alternative. Insertion sequencing (INSeq) is a method that enables production of targeted Tn mutant libraries and has been used successfully to identify L. pneumophila virulence phenotypes. In this chapter, a protocol is described for using INSeq to generate an arrayed L. pneumophila Tn mutant library and for subsequent screening of targeted mutant pools in a mouse model of infection.