Identification and Characterization of a Francisella tularensis Protein required for Vacuole Escape and Tularemia Development

The potential bioterrorism agent, Francisella tularensis ( F. tularensis ), causes the fatal disease tularemia with as few as 10 bacteria. Tularemia develops by bacteria invading, replicating, and spreading continuously within cells of the host. For a brief period of time following host cell invasion, F. tularensis occupies a membrane‐bound vacuole called a Francisella containing vacuole (FCV). Bacteria must break out of this vacuole to replicate in the host cytosol and spread from cell to cell. We tested the hypothesis that Francisella proteins responsible for escape from the vacuole are required for disease. To identify disease‐causing proteins within F. tularensis , we screened a mutant library of a commonly used surrogate microbe, F. tularensis subspecies novicida ( F. novicida ), and found that mutants for each of 195 genes were deficient in invasion and intracellular replication in cultured hepatocytes. To screen for proteins critical for tularemia development, we infected mice with a subset of mutants in 32 genes. We discovered disease was dramatically attenuated in mice infected with bacteria mutant for gene GS1a. Immunofluorescent and electron microscopic studies subsequently revealed that the mutant bacteria were trapped in large vacuoles. Our findings suggest that the protein GS1a is involved in bacterial escape from FCVs and is ultimately important for disease. Funded by CIHR and NSERC.