Differential expression of

Abstract

We have used RT-PCR and GFP-mediated fluorescence to analyse the regulation of PrfA-dependent virulence genes of Listeria monocytogenes during proliferation in mammalian host cells. Our data show that most of the PrfA-regulated virulence genes are more efficiently expressed, as measured by transcript levels, when L. monocytogenes is grown in macrophages and macrophage-like cells rather than in epithelial cells, hepatocytes or endothelial cells. The promoters for hly and plcA are predominantly activated within the phagosomal compartment, while those for actA and inlC are predominantly activated in the host cell cytosol. Expression of actA and plcB precedes that of inlC after infection of epithelial cells and macrophages. Little transcription of inlA or inlB is observed in epithelial cells and there is only slightly more in macrophages. In both cell types the level of transcription of the inlAB operon is lower than is seen under extracellular growth conditions in rich media, which is compatible with the assumption that InlA and InlB are not required during intracellular growth of the bacteria. Activation of the PrfA-independent iap promoter is also low during intracellular growth, although the gene product (p60) is required for cell viability. The levels of the PrfA-dependent virulence gene transcripts do not correlate with the amount of prfA transcript present, which is low under all intracellular conditions analysed, suggesting that the prfA transcript is either highly unstable in bacteria that are growing intracellularly, or that the small amount of PrfA produced is highly activated by additional component(s).

Microinjection and growth of bacteria in the cytosol of mammalian host cells

“…To distinguish between bacteria microinjected into the host cell cytosol and bacteria released into the culture medium during the microinjection, we took advantage of our recent observation (8) that the expression of the gfp cDNA (encoding the GFP) under the control of the promoter for the listerial virulence gene actA is strongly activated in the host cell cytosol. This PrfA-dependent promoter (P actA ) shows, however, very low activity when the bacteria are grown in rich culture media or reside in the phagosome of infected host cells (8,15). Hence, GFP-mediated fluorescence is expressed by listeriae that are injected into the host cell cytosol but not by those listeriae that are released during microinjection into the culture medium.…”

“…In L. monocytogenes, cytosolic replication seems to require the gene for a sugar phosphate transporter specifically controlled by the transcription factor, PrfA, which differentially regulates most of the known listerial virulence genes (1,2,15,19). PrfA is known to be active particularly when the bacteria grow in the cytosol of host cells (8,15), and the induced expression of this sugar uptake system by the host cell cytosol environment may enable L. monocytogenes to utilize glucose-1-phosphate (24), which is possibly derived from glycogen of the host cell. Similar uptake systems for phosphorylated sugars are also present in E. coli (27) and possibly other bacteria, but their expression in the cytosolic milieu of host cells may be less efficient than the PrfA-regulated uptake system of L. monocytogenes.…”

“…Strain EGD and its mutant derivatives were transformed by electroporation with the plasmid pLSV116-P actA -gfp (8, 15), pKSBC16-P sod -gfp (J. Daniels and A.B., unpublished data), or pJOE-P actA -gfp as described (8,15).…”

“…The amplification of the DNA sequence by PCR was performed in a 100-l reaction volume following standard protocols. This fragment was inserted into the PstI site of pLSV16-P actA -gfp (8), which resulted in the recombinant plasmid pJOE-P actA -gfp carrying hpt with its own promoter and gfp under the control of the actA promoter, P actA .Strain EGD and its mutant derivatives were transformed by electroporation with the plasmid pLSV116-P actA -gfp (8, 15), pKSBC16-P sod -gfp (J. Daniels and A.B., unpublished data), or pJOE-P actA -gfp as described (8,15).Microinjection Procedure. A total of 5-8 pl of a suspension of GFP-labeled bacteria in PBS buffer at a cell density of 10 6 bacteria per ml was microinjected into a single Caco-2 cell.…”

Hpt, a bacterial homolog of the microsomal glucose- 6-phosphate translocase, mediates rapid intracellular proliferation inListeria

“…The plasmid pPactA-gfp (14), carrying the gfp cDNA encoding the green fluorescent protein (GFP) under the control of the promoter of the listerial actA gene, was used to monitor the bacterial population present in the host cell cytosol. The actA promoter, strictly dependent on PrfA (see Fig.…”