• The Francisella tularensis Phagosomal Transporter Subfamily of Major Facilitator Superfamily Transporters is a Critical Determinant of Pathogenesis and Virulence and Modulates the Host Immune Response

      Marohn, Mark; Barry, Eileen M. (2012)
      Francisella tularensis (Ft) is the causative agent of tularemia. Due to its aerosolizable nature, high virulence, and low infectious dose, Ft poses a significant bioterror threat. It is classified as a category A select agent by the CDC and is a priority for therapeutics and vaccine development. Towards this goal, a greater understanding of its pathogenic mechanisms was investigated. The Ft genome is predicted to encode 31 Major Facilitator Superfamily (MFS) transporters, and the nine-member Ft phagosomal transporter (Fpt) subfamily possesses homology to virulence factors in other intracellular pathogens such as Legionella pneumophila. Intracellular survival and replication is critical to Ft pathogenesis. The central hypothesis tested was that these Fpt transporters are involved in normal intracellular survival and replication of Ft and host response to infection and will serve as viable targets for attenuation and vaccine development. Mutational analysis identified three fpt mutants (LVSδfptB, LVSδfptE, and LVSδfptG) that exhibited altered intracellular replication kinetics and attenuation of virulence in mice compared to wild-type Ft LVS. Vaccination of mice with a single dose of each mutant strain was protective against lethal intraperitoneal challenge. Like Ft LVS, these mutant strains colonized the lungs, liver and spleen of infected mice. However, there was often a delay in colonization and these strains did not replicate in the organs to the same extent as Ft LVS. The fpt mutants were completely cleared from the organs within 3-4 weeks after infection. While the cytokine responses to infection with these mutant strains were largely unaffected in murine macrophages when compared to Ft LVS, there were pronounced differences in cytokine profiles in the livers of infected mice. The altered in vivo cytokine responses may contribute to the attenuation observed in these strains. Future work will investigate the function of each of the transporters and the cellular and molecular mechanisms that underlie the attenuation of these strains. Overall, the data presented herein confirms the hypothesis that members of the Fpt family are critical factors in the pathogenesis of Ft and represent promising targets for development of efficacious live-attenuated vaccines.
    • Pediatric Cell-Mediated Immune System and Response to Ty21a Typhoid Vaccination Compared to Adults

      Rudolph, Mark Edward; Sztein, Marcelo B.; 0000-0002-0942-0349 (2018)
      Typhoid fever is a life-threatening disease caused by the human-restricted pathogen Salmonella enterica serovar Typhi (S. Typhi). The oral live-attenuated Ty21a typhoid vaccine protects against this severe disease by eliciting robust, multifunctional cell mediated immunity (CMI), shown to be associated with protection in wild-type S. Typhi challenge studies. Ty21a induces S. Typhi-responsive CD8+ and CD4+ T cells but little is known about the response to this vaccine in children. To address this important gap in knowledge, we have used mass cytometry to analyze pediatric and adult pre- and post-Ty21a vaccination T cells with both an HLA-E restricted and an autologous S. Typhi-antigen presentation model. Here, using conventional supervised analytical tools, we show adult T cells are more multifunctional at baseline than those obtained from children. Moreover, pediatric and adult T cells respond similarly to Ty21a vaccination, but adult responders remain more multifunctional. The use of the unsupervised dimensionality reduction tools allowed us to confirm these findings, as well as to identify increases and decreases in well-defined specific CD4+ and CD8+ T cell populations that were not possible to uncover using the conventional gating strategies. These findings evidenced age-associated maturation of multifunctional S. Typhi-responsive T cell populations, including those which have previously shown to be associated with protection from, and/or delayed onset of, typhoid disease. Further, in depth analysis of control stimulation conditions also found age-associated multifunctional T cell heterogeneity. These findings are likely to play an important role in improving pediatric vaccination strategies against S. Typhi and other enteric pathogens.