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Infection and Immunity, January 1999, p. 131-139, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Expression of Listeriolysin O and ActA by Intracellular and Extracellular Listeria monocytogenes

Marlena A. Moors,1,dagger Brian Levitt,1 Philip Youngman,2,Dagger and Daniel A. Portnoy1,*

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076,1 and Department of Genetics, University of Georgia, Athens, Georgia 306022

Received 27 July 1998/Returned for modification 18 September 1998/Accepted 15 October 1998

Listeria monocytogenes requires listeriolysin O (LLO) and ActA, the products of hly and actA, respectively, to establish a productive intracellular infection. LLO is essential for vacuolar lysis and entry into the cytosol, while ActA is required for bacterial spread to adjacent cells. We have used a transcriptional reporter gene system to compare the expression of actA and hly during intracellular growth to that during growth in broth cultures. The hly and actA genes were transcriptionally fused to Escherichia coli lacZ and Bacillus pumilus cat-86 (cat), and the fusions were integrated in single copies into the L. monocytogenes chromosome. A chloramphenicol resistance assay indicated that the hly fusion but not the actA fusion was significantly activated in Luria-Bertani (LB) broth, and this finding correlated with LLO and ActA levels detectable in broth cultures. Quantitation of promoter activity on the basis of beta -galactosidase activity revealed up to 10-fold-higher level of expression of the hly fusion relative to the actA fusion in LB broth. In contrast, both fusions were active in the cytosol of J774 cells, and the activity of the actA fusion was approximately 3-fold higher than that of the hly fusion under these conditions. However, quantitative immunoprecipitation of ActA and LLO from infected J774 cells demonstrated approximately 70-fold more cytosolic ActA than cytosolic LLO. Finally, in comparison to induction in broth cultures, actA was highly induced (226-fold) and hly was moderately induced (20-fold) in J774 cells. Collectively, these results indicate that actA and hly are differentially regulated in response to the growth environment and that both genes are preferentially expressed during intracellular growth. Further, while the lower level of production of ActA than of LLO in broth can be accounted for by transcriptional regulation, the relative abundance of intracellular ActA compared to that of intracellular LLO is a function of additional, possibly host-mediated, factors.

* Corresponding author. Present address: Department of Molecular and Cell Biology and The School of Public Health, University of California, Berkeley, CA 94720-3202. Phone: (510) 643-3925. Fax: (510) 643-5035. E-mail: portnoy{at}uclink4.berkeley.edu.

dagger Present address: Department of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, NC 27157.

Dagger Present address: Millennium Pharmaceuticals, Cambridge, MA 02139.

Infection and Immunity, January 1999, p. 131-139, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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