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Infection and Immunity, August 2009, p. 3244-3248, Vol. 77, No. 8
0019-9567/09/$08.00+0     doi:10.1128/IAI.00395-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Directed Mutagenesis of the Rickettsia prowazekii pld Gene Encoding Phospholipase D ^,

Lonnie O. Driskell,¹ Xue-jie Yu,² Lihong Zhang,² Yan Liu,^2,3 Vsevolod L. Popov,² David H. Walker,² Aimee M. Tucker,¹ and David O. Wood^1*

Department of Microbiology and Immunology, Laboratory of Molecular Biology, University of South Alabama College of Medicine, Mobile, Alabama 36688,¹ Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0609,² Anhui Province Center for Disease Control and Prevention, Hefei, Anhui Province, People's Republic of China³

Received 8 April 2009/ Returned for modification 21 April 2009/ Accepted 26 May 2009

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.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Laboratory of Molecular Biology, University of South Alabama, Mobile, AL 36688-0002. Phone: (251) 460-6324. Fax: (251) 460-7931. E-mail: dowood{at}jaguar1.usouthal.edu

Published ahead of print on 8 June 2009.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: R. P. Morrison

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Infection and Immunity, August 2009, p. 3244-3248, Vol. 77, No. 8
0019-9567/09/$08.00+0     doi:10.1128/IAI.00395-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.