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Pyrolysis gas-liquid chromatography (PGLC) was used to differentiate between HeLa cell-invasive and noninvasive strains of Yersinia enterocolitica and between Sereny-positive and -negative strains. A temperature-programmed gas-liquid chromatograph, equipped with a high-resolution Carbowax 20M coated capillary column, separated the volatiles from pyrolyzed whole cells preparations and cell wall fractions. The resulting pyrolysis elution patterns (pyrograms) were divided into 313 30-s time interval areas. The time interval areas were normalized in relation to the entire pyrogram area and were evaluated by stepwise linear discriminant analysis. The results of the PGLC-statistical analyses showed good correlation in prediction of the HeLa cell invasivity test. The technique of PGLC coupled with statistical analyses is objective, in contrast to traditional methods of determining pathogenicity of Y. enterocolitica.

Heat-stable enterotoxin (ST) of Yersinia enterocolitica was produced under defined conditions. It was first detected in the culture supernatant of the late-logarithmic phase of growth and increased lineally during the the stationary phase of growth. The ST level became maximum at the decline phase of growth, and the ST was not detected in the lysate of bacteria obtained from the decline phase of growth. The ST was extensively purified from the culture supernatant, and about a 1,905-fold purification was achieved with a yield of 8.9%. The minimal effective dose of the purified ST was approximately 25 ng in the suckling mouse assay. The purified ST gave a single 280-nm absorbing peak on polyacrylamide disc gel electrophoresis and had a maximum absorption at 272 nm, and its molecular weight was 9,700 by Sephadex G-75 superfine gel filtration. The biological activity of the purified ST was lost by treatment with 2-mercaptoethanol, suggesting that the ST contained disulfide bridges in the molecule which were required for the development of toxic activity. The purified ST was heat stable at 100 degrees C for 10 min between pH 2.2 and 8.0, but not at pH values greater than 9.0 or in 2 N HCl. The treatment of the ST with trypsin resulted in a retarded elution of the ST activity by Sephadex F-75 superfine gel filtration and a passage through a UM-20 membrane filter.

By using a suckling mouse assay, heat-stable enterotoxin (ST) was purified from the culture filtrate of Yersinia enterocolitica isolated from a diarrheal patient. The purification procedures involve ultrafiltration with an Amicon HIP-10 hollow fiber, ethanol fractionation, protamine sulfate treatment, diethylaminoethyl-Sephacel and hydroxylapatite column chromatographies, and Sephacryl S-200 superfine gel filtration. About 408-fold purification was achieved, with a yield of 12.0%. The minimal effective dose of purified ST was about 110 ng in the suckling mouse assay. The molecular weight of purified ST was 9,000 by Sephadex G-100 superfine gel filtration. The purified ST was stable to heating (100 degrees C for 20 min, 121 degrees C for 20 min) and did not lose its toxicity after treatment with protease, trypsin, lipase, phospholipase C, ribonuclease, deoxyribonuclease, beta-glucosidase, and neuraminidase. The purified ST was separated by isoelectric focusing into two active fractions, with pI's of 3.29 (ST-1) and 3.00 (ST-2), respectively. Antiserum from guinea pigs immunized with the purified ST neutralized the activity of both Y. enterocolitica ST and Escherichia coli ST.

The polysaccharide of the O-specific side chain of the lipopolysaccharide from the Yersinia pseudotuberculosis VA serovar has been isolated and studied. The structural pattern of the chemical repeating unit of the specific polysaccharide has been proposed as follows: (formula; see text).

Lipid A was isolated from lipopolysaccharide of Yersinia pseudotuberculosis S form (strain 341, subtype IB) using mild hydrolysis with acetic acid. The purified material (yield about 25%, molecular weight about 2900) contained D-glucosamine (11%), fatty acids (54%), protein concomitant (9.7%) and phosphorus (approximately 2%). Dodecanoic and 3-hydroxy-tetradecanoic acids in a molar ratio of 1 : 3.6 were detected as major fatty acid constituents. The hydroxyl groups of D-glucosamine were acylated with the residues of both fatty acids, while the amino groups were substituted with the residue of 3-hydroxy-tetradecanoic acid. Such a simple fatty acid composition is reminiscent of that found in lipid A in Y. pestis.

The chemical properties and the general biological activities of lipopolysaccharide (LPS) and Boivin-type endotoxin obtained respectively by phenol-water and trichloroacetic acid extraction from Yersinia enterocolitica serotypes O3 and O9 were studied. The yield of LPS from the O9 strain was about 10% of the O3 strain possibly because of the lower solubility of O9-LPS in aqueous phase. However, the chemical composition of O9-LPS was similar to that of O3-LPS in the proportions of reducing sugar, glucosamine, heptose, KDO, and lipid A. In pyrogenicity and local Shwartzman reactivity in rabbits and lethality for mice, there was also no difference between O3 and O9-LPS. The anthrone-positive carbohydrate and lipid A contents of Boivin-type endotoxin from O3 were higher than those of the endotoxin from O9. The biological activities of Boivin-type endotoxin from O3 were also remarkably higher than those of the endotoxin from O9. It seems that endotoxin of Y. enterocolitica serotype O3 may play an important role in infection by this organism.

A procedure is described for fractionating Yersinia grown in small cultures into inner and outer membranes and soluble cytoplasmic proteins. The procedure was applied to the three recognized species of the genus grown under conditions simulating mammalian intracellular fluid with respect to Ca2+ and Mg2+. These conditions are known to elicit the production of the plague virulence antigen V. Isolates capable of making this antigen were compared with virulence-antigen-negative derivatives by two-dimensional electrophoresis. The V antigen was localized to the soluble protein fraction as a peptide that comigrates with the major component of a specific immunoprecipitate. This peptide had an apparent molecular weight of 38,000 and was not found in either apparent molecular weight of 38,000 and was not found in either membrane fraction. The comparison of virulence antigen-producers and nonproducers of Y. pseudotuberculosis and Y. enterocolitica revealed large qualitative and quantitative differences in outer membrane protein patterns, whereas the same comparison for Y. pestis showed only minor differences. The complexity of changes in the various protein fractions corroborate data in the literature indicating that extensive physiological changes occur in virulent organisms cultivated under simulated intracellular conditions.

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.

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.

We have previously shown that under iron limitation, different Yersinia species synthesize new polypeptides. Two of them, the high-molecular-weight proteins (HMWPs), are expressed only by the highly pathogenic strains. In the present study, the HMWPs from Y. enterocolitica serovar O:8 were purified by gel filtration, and specific antibodies were obtained. Using these antibodies, we show that the two polypeptides were synthesized de novo during iron starvation and that they were found essentially in the bacterial outer membrane fractions, although the majority of the molecules were not exposed on the cell surface. We also demonstrate that the two proteins had common epitopes and that the HMWPs of the high-virulence-phenotype species Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serovar O:8 (a strain different from the one used to purify the proteins) are antigenically related. The less pathogenic and nonpathogenic strains did not exhibit cross-reacting material, suggesting that these strains do not synthesize even an altered form of the HMWPs.