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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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