title: Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes.
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 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.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Moors
authors.forename: B
authors.initials: B
authors.lastname: Levitt
authors.forename: P
authors.initials: P
authors.lastname: Youngman
authors.forename: D A
authors.initials: DA
authors.lastname: Portnoy
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001427:Bacterial Toxins; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002702:Chloramphenicol Resistance; D003470:Culture Media; D005110:Extracellular Space / Q000378:metabolism / Q000382:microbiology*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000138:chemical synthesis; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics; D007424:Intracellular Fluid / Q000378:metabolism / Q000382:microbiology*; D007763:Lac Operon; D008089:Listeria monocytogenes / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism / Q000382:microbiology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003470:Culture Media; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C072980:actA protein, Listeria monocytogenes; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; C038705:hlyA protein, Listeria monocytogenes