Development of genetic platforms for virulence gene identification and live vaccine generation in Francisella tularensis

Francisella tularensis is a facultative intracellular pathogen that causes the disease tularemia. F. tularensis is one of the most infectious bacteria known, with as few as 10 colony forming units able to cause disease in humans. Francisella is comprised of four subspecies, of which subspecies tularensis (type A) and subspecies holarctica (type B) are the biovars most commonly associated with disease in humans. Type A strains cause the most severe disease manifestations, particularly when acquired via the respiratory route. Pneumonic tularemia resulting from inhalation of aerosolized type A organisms can be highly debilitating, and has mortality rates approaching 30% to 60% if left untreated. Due to its low infectious dose and ease of dissemination, the United States Centers for Disease Control (CDC) has classified F. tularensis as a category A select agent, or one of the most likely agents to be utilized as a biological weapon. Currently, there is no licensed vaccine against F. tularensis infection. A live vaccine strain (LVS) was developed in the former Soviet Union from multiple passages of the less pathogenic type B biovar, but is unlicensed in the United States for a number of reasons, including an unknown basis of attenuation. Furthermore, LVS offers limited protection against respiratory challenge with type A strains, the most likely scenario in the event of a biological attack. Work leading to the development of new live vaccine candidates in Francisella has been hindered by a lack of useful genetic tools and a paucity of information regarding the genetic factors required for its pathogenesis. Very few vectors and selectable markers are available for use in F. tularensis species, and mutagenesis strategies for construction of defined genetic lesions have proven inefficient. These limitations have hampered the ability to construct defined mutations in highly virulent type A strains for generation of effective live vaccine candidates, as well as for the identification of genetic determinants and mechanisms utilized by F. tularensis for survival and replication in mammalian hosts. In the current study, we describe the development and utilization of genetic tools to address these concerns. We have utilized genetic platforms developed in our laboratory to construct defined, attenuating mutations in type A and B strains. The resulting strains were subsequently characterized in vitro and in vivo, and examined as potential live vaccines. Both strains were able to confer protective immunity in mice against subsequent lethal challenge with virulent Francisella. We also report the development of a global screen allowing for the identification of genes specifically expressed during growth of F. tularensis in murine macrophages or in mice. Using this strategy, we have identified a panel of Francisella genes specifically expressed during infection of murine J774A.1 macrophages. Collectively, the generation and optimization of genetic tools in our laboratory has allowed for construction and characterization of potential live vaccines, as well as the identification of genes potentially important to Francisella pathogenesis. These determinants contribute to the understanding of Francisella virulence, and may be exploited in the future for development of novel vaccines and/or therapeutics.