1887

Abstract

A novel plasmid for the analysis of promoter elements by site-specific integration into the genome of was constructed. The versatility of this reporter system was demonstrated by comparing the activity of the promoter in the high-pathogenic serotype O : 8 (strain WA-314) with that of the low pathogenic serotype O : 9 (strain Y127). The luciferase activity of a transcriptional fusion between the promoter of serotype O : 8 and was about fourfold lower than the activity of the respective O : 9 promoter. This correlated with lower invasin production by serotype O : 8 compared with serotypes O : 9, O : 3 and O : 5,27. However, of serotype O : 8 revealed higher invasiveness than serotype O : 9. When both invasins were expressed at similar levels in the O : 8 Δ background strain, cell invasion assays showed a slightly higher invasiveness of the strain producing Inv(O : 8) than the strain producing Inv(O : 9). We provide experimental evidence that this might be due to a higher binding capacity of Inv(O : 8) for cells expressing 1 integrins compared with Inv(O:9). The O : 8 strain harbouring the P  : :  fusion was then successfully used to follow expression in a mouse infection model. These experiments showed for the first time that the promoter is active in infected living mice, especially in Peyer's patches of the ileum, the caecal lymph follicle, and the lymph nodes, liver and spleen. The production of invasin in the spleen was demonstrated by Western blot analysis. In conclusion, the presented reporter system enables stable genomic integration of the operon into the chromosome of , facilitates quantification of promoter activities under different bacterial growth conditions, and enables detection of promoter activities in a mouse model.

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2010-09-01
2019-10-19
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