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Infection and Immunity, November 2008, p. 4934-4943, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00475-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

RipA, a Cytoplasmic Membrane Protein Conserved among Francisella Species, Is Required for Intracellular Survival{triangledown}

James R. Fuller, Robin R. Craven, Joshua D. Hall, Todd M. Kijek, Sharon Taft-Benz, and Thomas H. Kawula*

Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Received 16 April 2008/ Returned for modification 21 May 2008/ Accepted 24 August 2008

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS {Delta}ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS {Delta}ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS {Delta}ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, CB# 7290, 804 MEJB, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7290. Phone: (919) 966-9699. Fax: (919) 962-8103. E-mail: kawula{at}med.unc.edu

{triangledown} Published ahead of print on 2 September 2008.

Editor: S. R. Blanke

Infection and Immunity, November 2008, p. 4934-4943, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00475-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hall, J. D., Woolard, M. D., Gunn, B. M., Craven, R. R., Taft-Benz, S., Frelinger, J. A., Kawula, T. H. (2008). Infected-Host-Cell Repertoire and Cellular Response in the Lung following Inhalation of Francisella tularensis Schu S4, LVS, or U112. Infect. Immun. 76: 5843-5852 [Abstract] [Full Text]  


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