Title page for ETD etd-0702102-164050


Type of Document Master's Thesis
Author Mercante, Jeffrey W.
Author's Email Address jmerca2@lsu.edu
URN etd-0702102-164050
Title In-Vitro Gene Disruption Using Tn7-Based Transposition: Evaluation of Its Utility and Efficiency in Generating a Brucella VirB1 Knockout
Degree Master of Science (M.S.)
Department Microbiology (Biological Sciences)
Advisory Committee
Advisor Name Title
John R. Battista Committee Chair
Philip H. Elzer Committee Member
Vincent L. Wilson Committee Member
Keywords
  • cell free
  • facultative
  • phagocyte
  • macrophage
  • in-vitro
  • GPS-M
  • brucellosis
  • transposition
  • target immunity
  • PMN
  • antibiotic resistance
  • transposon
  • proteobacteria
  • gene inactivation
  • Brucella
  • kanamycin
  • abortus
  • Tn7
  • melitensis
  • polymorphonuclear
  • vaccine
  • Tn5
  • ruminant
  • gene disruption
  • virulence
  • attenuation
  • recombination
  • virB1
  • mutant
  • neutrophil
  • mutagenize
  • intracellular
  • pBBR1MCS
  • mouse
  • cattle
  • knockout
  • sheep
  • pathogen
  • pGPS3
  • pGEMT
  • mutagenesis
  • virB
  • in-vivo
  • goat
  • abortion
Date of Defense 2002-06-21
Availability unrestricted
Abstract
A Tn7-based transposition system carrying a kanamycin resistance gene has been used to disrupt a known virulence gene of Brucella melitensis 16M and Brucella abortus 2308 in-vitro. The suicide plasmid pBAvirB1::kan-29 was created, in part, by cloning the B. abortus virB1 gene into a vector that is not stably maintained in Brucella spp. The cloned virB1 gene was then mutagenized by in-vitro transposition of a Tn7-based transposon. This construct was used in-vivo to insert a disrupted virB1 gene in place of the wild type virB1 found in B. abortus 2308 and B. melitensis 16M by homologous recombination. The resulting virB1 mutant brucellae exhibited resistance to kanamycin, decreased survival in murine macrophages and attenuated virulence in a BALB/c mouse model. This method is proposed to be a simple, efficient means of generating gene knockouts in Brucella spp., and may be useful in targeting virulence factors for the creation of live, attenuated vaccines. The genus Brucella consists of bacteria that are facultative intracellular pathogens. Individuals in this genus of Gram-negative coccobacilli are responsible for causing late term spontaneous abortion in cattle and goats as well as the human disease brucellosis (undulant fever). The success of Brucella is dependent on their ability to invade, survive and multiply within phagocytes. The virB operon, consisting of eleven genes in most Brucella species, is thought to contribute to a type IV secretion system responsible for maintaining infection in a mammalian host. Further results of this study suggest that the Brucella virB1 gene product, like its Agrobacterium counterpart, may not be essential for virulence; disruption of the Brucella virB1 gene results in a one to two log decrease in intracellular survival at forty-eight hours in an in-vitro macrophage model and by four weeks in an in-vivo mouse model.
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