InSeq analysis of defined libraries identifies a transporter-encoding gene cluster important for intracellular replication in mammalian hosts.

is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit for infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria.