Analysis of Yersinia enterocolitica invasin expression in vitro and in vivo using a novel luxCDABE reporter system.
A novel luxCDABE plasmid for the analysis of promoter elements by site-specific integration into the genome of Yersinia enterocolitica was constructed. The versatility of this reporter system was demonstrated by comparing the activity of the inv promoter in the Y. enterocolitica 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 inv promoter of serotype O : 8 and luxCDABE was about fourfold lower than the activity of the respective O : 9 promoter. This correlated with lower invasin production by Y. enterocolitica serotype O : 8 compared with serotypes O : 9, O : 3 and O : 5,27. However, Y. enterocolitica of serotype O : 8 revealed higher invasiveness than serotype O : 9. When both invasins were expressed in trans at similar levels in the Y. enterocolitica O : 8 Deltainv 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 beta1 integrins compared with Inv(O:9). The Y. enterocolitica O : 8 strain harbouring the P(inv)((O : 8)) : : luxCDABE fusion was then successfully used to follow inv expression in a mouse infection model. These experiments showed for the first time that the inv 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 luxCDABE operon into the chromosome of Yersinia, facilitates in vitro quantification of promoter activities under different bacterial growth conditions, and enables detection of promoter activities in a mouse model.
Trček
Janja
J
Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany.
Fuchs
Thilo M
TM
Zentralinstitut für Ernährung- und Lebensmittelforschung, Abteilung Mikrobiologie, Technische Universität München, Germany.
Trülzsch
Konrad
K
Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany.
D018829:Adhesins, Bacterial / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014774:Virulence; D015009:Yersinia Infections / Q000382:microbiology; D015008:Yersinia enterocolitica / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity
D018829:Adhesins, Bacterial; C053476:invasin, Yersinia; D008156:Luciferases