F tularensis host (lung) immune responses, genetics of virulence
Program leader: Jeff Frelinger
SE-RP-008: Route of Infection Shapes Immune Responses to Francisella
Jeff Frelinger
University of Arizona
University of North Carolina
The goal of this project is to understand how the route of infection by Francisella tularensis is shapes the subsequent adaptive immune response. In order to do this we have identified the infected cells in the lung following intranasal inoculation and in the skin following intradermal inoculation. In the lung the first cells infected are the alveolar macrophages, not interstitial macrophages. In contrast the cells infected in the skin are all found in the dermis and are not Langerhans cells but are tissue macrophages. Thus, the cell types initially infected differ in the two routes of infection. Current work focuses on purifying sufficient numbers of cells from both organs to permit the determination of the cytokine profiles following infection and comparing them to the expression patterns from bone marrow derived macrophages which are the most commonly used cells for analysis.
SE-RP-009: Molecular characterization of F. tularensis SchuS4 virulence factors
Emory University
James Bina and Mark Miller
University of Tennessee at Memphis
Francisella tularensis is a highly infectious Category A bacterial pathogen that causes tularemia, a potentially life-threatening disease in humans. Due to the ease of aerosol dissemination of this organism and the minimal inoculum (≤10 bacteria) necessary to cause severe disease, F. tularensis has been weaponized for use in biowarfare. Critical to Francisella’s pathogenesis are its ability to replicate within macrophages, the primary niche for replication in vivo, and to subvert the host immune response. Unfortunately, relatively little is known about which genes F. tularensis uses to subvert host defenses and how these genes are regulated. We recently employed a powerful global in vivo negative selection screen in mice to identify genes required for the pathogenesis of F. novicida, a subspecies of Francisella that causes disease in mice but not humans. This approach resulted in the identification of 164 genes that are required for virulence, 44 of which appear to encode novel virulence factors.
To further understand how these 164 genes contribute to Francisella pathogenesis, we have screened mutants in each gene for the ability to replicate in murine macrophages. This secondary screen revealed that 65 (~40%) of the 164 genes were required for replication in macrophages in vitro. These genes included those within the Francisella pathogenicity island (FPI), a critical virulence locus known to be essential for intracellular replication, thus validating the screen. One gene identified by the screen encodes a novel lipase which is critical for Francisella escape from the macrophage phagosome to the cytosol, where the bacteria replicate. We have now determined that this lipase contributes to expression of the FPI genes, a previously unappreciated and novel level of regulation of these critical virulence genes.
As a parallel approach to identify F. tularensis virulence genes, we have performed the negative selection screen in this highly virulent organism. This has allowed us to identify a “core set” of genes required for the pathogenesis of both F. novicida and F. tularensis, as well as to identify F. tularensis virulence genes not encoded in the less virulent Francisella subspecies. Elucidation of the functions of these genes may lead to an understanding of the enhanced virulence of F. tularensis. Taken together, our work will significantly enhance our understanding of Francisella pathogenesis as well as common themes in host-pathogen interactions, and help foster the development of the next generation of therapeutics and vaccines against Francisella.
