F tularensis host (lung) immune responses, genetics of virulence
Program leader: Jeff Frelinger
SE-RP-008: Route of Infection Shapes Immune Responses to Francisella
University of North Carolina
Bacterial pathogens are intimately involved with their hosts. Our efforts are focused on altering the host response in ways that are advantageous to the pathogen. In Francisella infection via the airway, the immune response in the lung is blunted for IFN-g and enhanced for IL-17. This is in contrast to the immune response in the lung following intradermal infection, even though the bacteria rapidly disseminate to the lung. Our hypothesis is that infection of particular cells in the lung results in a different pattern of host derived immunomodulatory molecules produced that shape the host innate and adaptive immune responses to benefit the pathogen. In this study we will determine the early cells infected in lung and skin infections. We will determine the molecules produced in the earliest cells following infection using a combination of fluorescence activated cell sorting and marked bacteria. Using cell purification and in vitro infection we will learn how products of infected cells can impact the subsequent immune response.
Narrative
Microbes and hosts have a complex interaction. Signals go from the microbe to the host and vice versa. We wish to understand how the bacterium that causes tularemia manages to send different signal to the host depending on where the initial infection occurs. We will identify the first cells infected by the bacteria and how they respond depending on whether infection happens in the lungs or the skin. We will then determine how these cell influence the outcome of the immune response to the bacterium, and which bacteria genes are required. This will allow us to define targets for intervention by drugs.
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. Many genes contribute to the ability of bacterial pathogens to replicate within the host, but distinct virulence factors, which are not required for replication, often play crucial roles in evading host defenses. 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 (called Transposon Site Hybridization or TraSH) in mice to identify genes required for the pathogenesis of F. novicida (1), a subspecies of Francisella that causes disease in mice but not humans (2). 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 individually 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) as well as other genes known to contribute to replication within macrophages, thus validating the screen. We are now pursuing the mutants with the most severe replication defects to determine how the proteins they lack contribute to Francisella pathogenesis at the molecular level. Elucidation of the molecular mechanisms of action of critical Francisella virulence factors will significantly enhance our understanding of Francisella pathogenesis as well as common themes in host-pathogen interactions. This work will generate data that will lay the groundwork for the next generation of therapeutics and vaccines against potential biowarfare agents and emerging infections.
