Francisella tularensis was a facultative intracellular pathogen and a gram-negative coccobacillus which has been categorized by the CDC as a potential class A select agent due to its highly infectious properties and high mortality rates. Francisella tularensis was also responsible for the zoonotic disease tularemia, which was usually transmitted by arthropod vectors or via contact with infected animals. Francisella tularensis subspecies novicida has been used by many researchers in genetic pathogenesis experiments to try to elucidate genes responsible for virulence factors. One of these virulence factors was a capsular material which has been thought to be involved in either increasing pathogenicity or infectivity of this organism upon engulfment by its principal host cell, the macrophage. There were many potential genetic loci which may be involved in this biosynthetic process of encapsulation. One such locus has excellent homology to the capsule biosynthesis operon of Bacillus anthracis, which, under certain conditions, creates a polyglutamic acid capsule (PGA). A transposon mutation in the amide ligase (capB) in LVS has a reduced virulence in murine infection models. I wished to investigate whether Francisella novicida was capable of producing such a capsule and under which environmental conditions this capsule was made. I have created a site-directed mutant of the capB gene in Francisella novicida U112 using targeted mutagenesis via PCR SOEing and have introduced this mutation via electroporation of a suicide vector. I have tested our mutant against preimmune serum treatments and have shown reduced viability as well as a reduced capacity for replication inside RAW 264.7 murine macrophages. I assayed for production of a PGA capsule via immunodot blot and electron microscopy as well as analysis by mass spectrophotometry of capsular extracts. I also tested various media constituents and different environmental conditions to determine which external stimuli may contribute to PGA capsule biosynthesis as well as regulatory changes in transcript levels of this operon.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1015 |
| Date | 28 July 2009 |
| Creators | Fleming, Eric |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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