Temperature-inducible and Calcium-regulated Proteins Encoded by the Virulence Plasmid of Yersinia Akademisk Avhandling

Temperature-inducible and calcium-regulated proteins encoded by the virulence plasmid of Yersinia. Ingrid Bölin, Department of Cellbiology and Microbiology, National Defence Research Institute, S-901 82 Umeå, Sweden. The pathogenic members of the genus Yersinia, Y. pseudotuberculosis, Y. pestis and Y. enterocolitica are transmitted from animals to man and may give rise to disease with ä~ variety of symptoms. These bacteria possess related plasmids necessary for virulence. In this study, gene products encoded by the virulence plasmid have been identified and characterized. A temperature-inducible outer membrane protein YOPl, is encoded by the virulence plasmid. YOPl is expressed by Y. pseudotuberculosis and Y. enterocolitica at 37°C. The genetic locale of trie structural gene for YOPl on the virulence plasmid was determined. A mutant that was unable to express this protein, remained fully virulent, showing that YOPl is not a virulence determinant. Several other proteins encoded+by the virulence plasmid are induced at 37°C in a medium lacking Ca . T^ese proteins are not expressed at 26°C and expression is repressed by Ca -concentrations in excess of 2.5 mM. In Ca -deficient medium, the induced proteins can be found extracellularly as well as in the outer membrane. However, in the presence of Ca at 37°C they are only found in the outer membrane. The released proteins consist of eight polypeptides as revealed by two-dimensional electro­ phoresis. These proteins, Y0P2a and 2b, YQP3, Y0P4a and 4b, the V-antigen and a small uncharacterized polypeptide, are expressed by all three pathogenic Yersinia species, both in vivo and in vitro. The Ca^+-controlled expression of the YOP proteins is regulated by genes in the Ca -region, which are conserved in the three species. Mutations in this region repress the expression of the Ca -regulated YOPs. The genetic loci identified for five of these proteins revealed that only the structural gene of the Y0P4b protein is part of the Ca -region. The other genes were found at separate locations outside this region. The structural genes for YOP4b, YOP3 and the V-antigen, together with the genes for two additional polypeptides, were localized to a common region conserved on the plasmids of the Yersinia species. The structural genes for Y0P2b (yopH) and Y0P5 (yopE) are located in different positions on the plasmid from Y. enterocolitica, compared to the other two species. This plasmid has Been rearranged so that these genes are located close to one another. The DNA sequence of the yopH gene shows that it is a singly transcrip­ tional unit. Transcription of this gene is regulated by Ca -concentra­ tion and by temperature. A mutant strain of Y. pseudo tuberculosis, de­ leted for the yopH gene on the virulence plasmid, is avirulent In mice. Virulence is restored by trans-complementation with the cloned yopH gene. The mutant strain is also’ unable to inhibit phagocytosis of macrophages as compared to the wild-type strain. The trans-compleroented strain shows inhibition comparable to that of the wild-type. Therefore, the YOP2b protein is considered to be an essential virulence determinant.