Affiliation:
1. Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076,1 and
2. Department of Genetics, University of Georgia, Athens, Georgia 306022
Abstract
ABSTRACT
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 β-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.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Immunology,Microbiology,Parasitology
Cited by
153 articles.
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