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Journal of Bacteriology, November 2002, p. 5935-5945, Vol. 184, No. 21
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.21.5935-5945.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Inducible Control of Virulence Gene Expression in Listeria monocytogenes: Temporal Requirement of Listeriolysin O during Intracellular Infection

Christina E. Dancz,1 Andrea Haraga,2,{dagger} Daniel A. Portnoy,2,3 and Darren E. Higgins1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115-6092,1 Department of Molecular and Cell Biology,2 School of Public Health, University of California, Berkeley, California 94720-32023

Received 2 May 2002/ Accepted 16 August 2002

We have constructed a lac repressor/operator-based system to tightly regulate expression of bacterial genes during intracellular infection by Listeria monocytogenes. An L. monocytogenes strain was constructed in which expression of listeriolysin O was placed under the inducible control of an isopropyl-ß-D-thiogalactopyranoside (IPTG)-dependent promoter. Listeriolysin O (LLO) is a pore-forming cytolysin that mediates lysis of L. monocytogenes-containing phagosomes. Using hemolytic-activity assays and Western blot analysis, we demonstrated dose-dependent IPTG induction of LLO during growth in broth culture. Moreover, intracellular growth of the inducible-LLO (iLLO) strain in the macrophage-like cell line J774 was strictly dependent upon IPTG. We have further shown that iLLO bacteria trapped within primary phagocytic vacuoles can be induced to escape into the cytosol following addition of IPTG to the cell culture medium, thus yielding the ability to control bacterial escape from the phagosome and the initiation of intracellular growth. Using the iLLO strain in plaque-forming assays, we demonstrated an additional requirement for LLO in facilitating cell-to-cell spread in L2 fibroblasts, a nonprofessional phagocytic cell line. Furthermore, the efficiency of cell-to-cell spread of iLLO bacteria in L2 cells was IPTG dose dependent. The potential use of this system for determining the temporal requirements of additional virulence determinants of intracellular pathogenesis is discussed.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115-6092. Phone: (617) 432-4156. Fax: (617) 738-7664. E-mail: dhiggins{at}hms.harvard.edu.

{dagger} Present address: Department of Microbiology, University of Washington, Seattle, WA 98195-7710.

Journal of Bacteriology, November 2002, p. 5935-5945, Vol. 184, No. 21
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.21.5935-5945.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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