Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D

Lonnie O. Driskell, Xue Jie Yu, Lihong Zhang, Yan Liu, Vsevolod Popov, David Walker, Aimee M. Tucker, David O. Wood

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

Original languageEnglish (US)
Pages (from-to)3244-3248
Number of pages5
JournalInfection and Immunity
Volume77
Issue number8
DOIs
StatePublished - Aug 2009

Fingerprint

Rickettsia prowazekii
Phospholipase D
Mutagenesis
Genes
Epidemic Louse-Borne Typhus
Virulence
Bacteria
Attenuated Vaccines
Homologous Recombination
DNA
Feasibility Studies
Southern Blotting
Site-Directed Mutagenesis
Life Style
Immunity
Guinea Pigs
Plasmids
Vaccines
Polymerase Chain Reaction
Mutation

ASJC Scopus subject areas

  • Immunology
  • Microbiology
  • Parasitology
  • Infectious Diseases

Cite this

Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D. / Driskell, Lonnie O.; Yu, Xue Jie; Zhang, Lihong; Liu, Yan; Popov, Vsevolod; Walker, David; Tucker, Aimee M.; Wood, David O.

In: Infection and Immunity, Vol. 77, No. 8, 08.2009, p. 3244-3248.

Research output: Contribution to journalArticle

Driskell, Lonnie O. ; Yu, Xue Jie ; Zhang, Lihong ; Liu, Yan ; Popov, Vsevolod ; Walker, David ; Tucker, Aimee M. ; Wood, David O. / Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D. In: Infection and Immunity. 2009 ; Vol. 77, No. 8. pp. 3244-3248.
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