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The structural genes of the outer membrane polypeptides of Yersinia spp. (YOPs) and the V antigen of plasmid pIB1 of Yersinia pseudotuberculosis were recently cloned and mapped (A. Forsberg, I. Bölin, L. Norlander, and H. Wolf-Watz, Microb. Pathogen. 2:123-137, 1987). The corresponding genes were localized on pYV019 and pYV8081 of Yersinia pestis and Yersinia enterocolitica, respectively. No obvious differences were observed on comparison of pIB1 and pYV019, whereas pYV8081 showed intragenic as well as extragenic changes. However, one region of plasmid pYV8081, which coded for the V antigen, YOP3, and YOP4a, was essentially conserved among the three plasmids. Since this region is connected with the Ca2+ region, we suggest that the conserved region of the virulence plasmids of Yersinia spp. be extended to include both of these regions. Low amounts of the YOPs were detected in the membrane fraction at 37 degrees C in the presence of 2.5 mM calcium. Only minor differences were noticed when the individual YOPs of Y. pestis and Y. pseudotuberculosis were compared. Several differences were observed when the YOPs of Y. enterocolitica were included for comparison. All Y. enterocolitica proteins, except YOP1, YOP4b, and the V antigen, exhibited changes in their characteristic molecular sizes. Although these differences were within a range of +/- 2 kilodaltons, the isoelectric point was retained for each protein type.

After tularemia vaccinations, most individuals respond with cell-mediated and humoral immunity as disclosed by the lymphocyte stimulation test and enzyme-linked immunosorbent assay (ELISA), respectively. There is, however, no correlation between the magnitudes of the two responses, and some individuals show one of the responses only. We now report that the two responses are directed towards different antigenic determinants of the bacterium. Ether-water extraction of the live vaccine strain of Francisella tularensis gave a high yield of material reacting in ELISA as well as in the lymphocyte stimulation test. However, the specificity of the extract was low insofar as it reacted not only with lymphocytes and antibodies of tularemia-vaccinated individuals but also to a fairly high extent with those of nonvaccinated individuals. By using the extract as a starting material, a preparatory procedure was developed, resulting in antigen of high specificity in the two tests. The antigen prepared was a high-molecular-weight, carbohydrate-protein complex. Proteinase K treatment of the antigen abolished the lymphocyte-stimulating activity but did not decrease ELISA activity at all. Periodate treatment, on the other hand, greatly reduced ELISA activity but did not decrease the lymphocyte-stimulating activity. Thus, determinants of F. tularensis responsible for immunospecific lymphocyte stimulation seem to reside in protein, whereas ELISA activity seems to be due mostly to carbohydrate determinants.

Virulence plasmid-containing cells of Yersinia pseudotuberculosis had the ability to inhibit phagocytosis by mouse peritoneal macrophages cultured in vitro, but cells of its plasmid-cured derivative did not. Inhibition was most pronounced when the pathogen was incubated under Ca2+-deficient conditions, which allowed a high level of expression of outer membrane proteins (Yops). The addition of 2.5 mM Ca2+ to the growth medium reduced the degree of inhibition by the pathogen, but it was still significantly higher than that of the plasmid-cured strain. An avirulent mutant strain, from which the entire yopH gene was deleted, was impaired in its phagocytosis inhibition ability. This mutant could be trans-complemented by the yopH+ gene back to the wild-type phenotype with respect to virulence, as well as the ability to inhibit phagocytosis, demonstrating that the ability to inhibit phagocytosis is an important virulence function. The mutant strain was still cytotoxic for HeLa cells, indicating that inhibition of phagocytosis can be genetically separated from the ability to cause a cytotoxic effect.

A strain of Yersinia pseudotuberculosis which harbors a 63-kilobase plasmid was found to cause a lethal infection in Swiss albino mice. The rate of infection paralleled the ability of the pathogenic organism to attach to a monolayer of HeLa cells. One novel outer membrane protein (protein 1) with a molecular weight of 140,000 was found to be associated with the possession of the 63-kilobase plasmid not at 26 degrees C, and expression was moderately affected by the concentration of calcium in the growth medium. Moreover, it was found that synthesis of protein 1 associated outer membrane protein showing similar properties was also found to be expressed in plasmid-containing strains of Yersinia enterocolitica. The properties of protein 1 indicate that it could be identical to the previously described virulence W antigen.

The expression of the temperature-inducible plasmid-coded outer membrane proteins (YOPs) of Yersinia pseudotuberculosis was studied. These proteins were not recovered in the outer membrane fraction when the strain was grown in minimal medium at 37 degrees C, but they were expressed under these conditions. A strict correlation was found between Ca2+ dependency in the virulent strain, YPIII(pIB1), and ability to express YOPs. Ca2+-independent plasmid mutants or RNA-polymerase mutants harboring the virulence plasmid were unable to express YOPs, in contrast to the wild-type strain. These strains were also found to be avirulent. Sera recovered from patients or animals undergoing infection with either Y. pseudotuberculosis, Y. pestis, or Y. enterocolitica possessed antibodies directed against YOPs, indicating that they were expressed in all three pathogenic Yersinia species during infection. The YOPs of the three different species showed high immunological relatedness.

The virulence plasmids pYV019, pYV8081, and pIB1 from Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis, respectively, were characterized by restriction endonuclease analysis. The three plasmids exhibited a region of common DNA previously shown to encode determinants which confer Ca2+ dependence. The plasmids from Y. pestis and Y. pseudotuberculosis were similar throughout their genomes. In contrast, a region of the plasmid from Y. enterocolitica which contained an origin of replication differed from the other two plasmids as determined by DNA homology and replication properties. Plasmid-associated outer membrane proteins from all three species of Yersinia were characterized by polyacrylamide gel electrophoresis. There were no differences in the outer membrane protein profiles between plasmid-containing and homogenic strains lacking the plasmid after growth at 28 degrees C. After growth at 37 degrees C, both Y. enterocolitica and Y. pseudotuberculosis showed at least four major plasmid-associated outer membrane proteins. Y. pestis did not show any discernible changes after growth at 37 degrees C. It was shown by using E. coli minicell analysis that the plasmid DNA from all three species of Yersinia contained the coding capacity for production of the novel outer membrane proteins.

When Yersinia pseudotuberculosis is shifted from growth at 26 degrees C to growth at 37 degrees C, the synthesis of a plasmid-associated outer membrane protein, protein 1, is induced (Bölin et al., Infect. Immun. 37:506-512). The structural gene of this protein was found to be located on the virulence plasmid pIB1 of Y. pseudotuberculosis. One cosmid hybrid plasmid pBW8 was studied which carried a region of the virulence plasmid. This hybrid plasmid expressed in Escherichia coli K-12 a novel temperature-inducible outer membrane protein which is immunologically related to and has the same molecular weight as protein 1. Protein 1 was purified to homogeneity, and 14 amino acids of the N-terminal end were determined. From this sequence, the tentative corresponding DNA sequence was deduced, and a set of 11-nucleotide-long DNA probes was chemically synthesized. By using these probes in Southern blotting experiments, the genetic location of the N-terminal end of protein 1 was established. By introducing the transposon Tn5 into the virulence plasmid pIB1, mutants were obtained that did not express protein 1. One class of these mutants was still Ca2+ dependent and virulent, suggesting that protein 1 is not a major virulence determinant. Tn5-derived insertion mutants were also obtained which were Ca2+ independent. Such mutants were found to be avirulent. One Ca2+-independent mutant still expressed protein 1, indicating that the regulatory expression of protein 1 is not linked to Ca2+ dependence.