Date of Degree
PhD (Doctor of Philosophy)
Bradley D. Jones
Francisella tularensis is a Gram-negative pathogenic organism that causes the disease tularemia. This disease can be potentially fatal without treatment. Francisella tularensis virulent strains can cause disease in humans with an infectious dose as low as 10 organisms. As a result of this low infectious dose, high mortality, and ease to produce an aerosol inoculum, the Centers for Disease Control and Prevention has classified Francisella tularensis as a Tier I select agent, the highest threat level. Much research has been done to determine the cause for the extreme virulence. However, despite these efforts, little is known about the mechanisms by which Francisella goes undetected inside host cells until it is too late for the host to respond. Researchers in the Jones' laboratory utilized a transposon site hybridization (TraSH) screen with human monocyte derived macrophages (MDMs) as the host cell and an enzyme-linked immunosorbent assay (ELISA) screen of pools of transposon mutants searching for virulence determinants and genes responsible for Francisella capsule or LPS. Through the TraSH screen, our group identified a locus of genes, FTT1236, FTT1237, and FTT1238c as being important for survival within human MDMs. From the mutant library screen using ELISA, I identified the same genes, FTT1236 and FTT1238c. In addition, I also identified wzy, wbtA, FTT0846, and hemH as being involved in LPS and or capsule production. A similar ELISA screen was done by researchers in Apicella laboratory using a different monoclonal antibody that identified insertions in, dnaJ, manB and an intergenic region between FTT0673 and FTT0674c that potentially disrupted LPS and capsule biogenesis. Previously, FTT1236, FTT1237, and FTT1238c mutants were observed by our laboratory to be serum sensitive and activate MDMs by an unknown mechanism. I further characterized these mutant strains by analyzing the changes in the LPS core. I identified core truncations for the FTT1236 and FTT1237 mutants, but not FTT1238c. Combining this new data with previously published work and bioinformatical analysis of the FTT1236, FTT1237 and FTT1238c proteins, I hypothesized that these proteins have functions similar to Waa proteins of other organisms, which are involved in LPS core assembly and O-antigen ligation. With functional complementation and mass spectrometry of LPS preparations, I have designated FTT1236, FTT1237, and FTT1238c as WaaY, WaaZ, and WaaL respectively. In addition to this work characterizing the biochemical functions of these gene products, I examined the effect of mutations in these genes on the virulence of Francisella. In contrast to infection with wild type Schu S4, mice infected either intraperitoneally or intranasally displayed significant inflammatory responses to infection and the strains were significantly attenuated by either route of infection. I also observed that waaY and waaL mutant strains disseminated to the liver and spleen after an intranasal infection despite their lack of O-antigen and capsule. At an i.n. dose of 106 CFU these mutant strains still caused lethal murine infection, but death occurred around day 12 post infection; mice infected with <20 CFU of Schu S4 succumb at day 5 post infection. The cause of the death in mice infected with these mutant strains was pulmonary edema, rather than multiple organ failure induced by Schu S4. Of the additional seven mutant strains identified from the ELISA screens, I characterized their physical phenotypes, virulence defects, and their potential as an attenuated live vaccine. All of these strains were determined to be sensitive to human pooled serum to various degrees. Three of these strains, dnaJ::Tn5, hemH::Tn5, and FTT0673p/prsAp::Tn5 did not have identifiable defects in capsule or LPS biosynthesis, nor were they attenuated in mice. The remaining four strains, FTT0846::Tn5, manB::Tn5, wzy::Tn5, and wbtA::Tn5, were found to have LPS O-antigen and capsule defects, and two of these strains had LPS core defects (FTT0846::Tn5 and manB::Tn5). Each of these four strains was attenuated in mice, when compared to WT. I also tested the ability of mice infected with waaY::TrgTn, waaL::TrgTn, and wbt::Tn5 to be protected from lethal challenges of Schu S4. All three strains provided some level of protection against lethal Schu S4 challenges. In addition, I also tested Francisella LPS and capsule to provide protection against lethal challenges of LVS and Schu S4. I determined that LPS and capsule protected against high doses of LVS, but LPS did not provide any protection when immunized mice were challenged with Schu S4. Interestingly, we observed that mice immunized with capsule were partially protected from lethal Schu S4 challenges. In addition, I observed a novel difference between virulent Francisella strains and LVS, in that virulent strains have O-antigen glycosylated and LVS appears to be lacking this characteristic. Collectively, this work adds to the growing data of the importance of LPS and the role of capsule role in immune evasion as well as the significance of capsule and LPS mutant strains to provide protection against Schu S4.
capsule, Francisella, LPS, vaccination
xvii, 152 pages
Includes bibliographical references (pages 134-152).
Copyright 2014 Jed Anthony Rasmussen