Graduate Thesis Or Dissertation


Developing a system of mutagenesis in Francisella tularensis LVS Public Deposited

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  • Francisella tularensis is a gram-negative facultative intracellular coccobacillus that primarily infects macrophages. The causative agent of tularemia, this bacterium is considered among the most infectious organisms known, requiring fewer than ten organisms to cause disease. Although ubiquitous in nature, transmission to humans is rare but can occur via insect bites, direct contact with infected animals, ingestion of contaminated water, or through the inhalation of aerosols. There are several species of Francisella, however the majority of infections are caused by F. tularensis. Species of F. tularensis are further classified into two groups according to pathogenesis, the Type A highly virulent strain and the Type B, less pathogenic strains. Type A pathogens cause a variety of clinical manifestations including several glandular infections and the most life threatening, pneumonic tularemia. Given the many routes of transmission, low infectious dose and severity of the illness, F. tularensis has become a concern for potential development of the bacteria into a bioweapon and has been classified as a Category A pathogen by the Centers for Disease Control and Prevention (CDC). Previous studies have attempted to investigate the pathogenicity of F. tularensis using a variety of genetic manipulation techniques. However due to the unique challenges of applying current genetic techniques in F. tularensis, few genes important for Francisella virulence have been identified. This study aims to develop a random transposon mutagenesis library and primary screening assay to rapidly identify virulence factors associated with intra-macrophage survival. A potential library was generated using plasmid pFT-mariner, a Francisella mutagenesis vector constructed for this study. This plasmid utilizes a eukaryotic mariner himar-1 transposase and transposon cassette. An arabinose inducible promoter that regulates transposase activity, controls transposition of the kanamycin flanked transposon cassette. The pFT-mariner plasmid was introduced into F. tularensis live vaccine strain (LVS) through conjugation and resulted in several potential library founder clones. Founder clones were screened by polymerase chain reaction (PCR) and found to contain pFT-mariner components in several generations of passed bacteria. Select clones were incubated with arabinose to induce transposon integration into the genome. A counter-selection method was used to eliminate the pFT-mariner plasmid. DNA from potential library clones was screened by PCR to detect the integration of the transposon and to verify the loss of the remaining plasmid. Following confirmation of transposition, several methods were used to try to determine the site of insertion. To screen for pathogenicity, any identified mutants would be applied to a macrophage infection assay and compared to a F. tularensis LVS infection. This study generated multiple potential library founder clones and developed a rapid screening assay for intra-macrophage survival of F. tularensis LVS. However in our investigation we encountered several difficulties; while we were able to detect transposon integration immediately following transposase induction, these failed to be identified again in subsequent investigation. Ultimately, similar to previously reported mutagenesis attempts our potential library of transposon mutants was determined to be unstable. Thus, future transposon mutagenesis efforts should focus on verifying stability of the vector and transposon.
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