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ABSTRACTTwo almost-identical strains of Eubacterium aerofaciensisolated from the normal human gut flora were used. The cell wall (CW) of one strain with a peptidoglycan (PG) type A4α induces chronic arthritis in the rat after a single intraperitoneal injection, whereas CW of the other with PG type A4β induces only a transient acute arthritis. The CW of the arthritogenic E. aerofacienswas a twofold-more-potent stimulator of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) than the nonarthritogenic CW. After degradation with mutanolysin, the capacity of the arthritogenic PG to stimulate production of TNF-α and MCP-1 was significantly increased, whereas that of the nonarthritogenic PG was significantly decreased. In other words, after enzyme degradation the arthritogenic PG had a four- to fivefold-stronger stimulatory capacity than that of the enzyme-treated nonarthritogenic PG. These findings indicate that the arthritogenicity of CW or a PG is not dependent on the enzyme resistance alone but also on how the PG fragments released by enzyme degradation stimulate the production of proinflammatory cytokines.

ABSTRACTVery little is known about how the host genome influences the composition of the gastrointestinal flora, largely due to the great number and diversity of bacteria present in the flora and the difficulties of using traditional methods of bacterial isolation and identification. We have approached the problem by studying bacterium-derived cellular fatty acids in the stool samples of six mouse strains congenic for the major histocompatibility complex (MHC). The results obtained indicate that the composition of the fecal flora is genetically regulated. In addition to undefined gene loci, MHC alone has a pronounced effect, since mice with different MHC in the same background have significantly different fecal floras. Demonstration of the genetic influence on the gastrointestinal flora opens a new approach to studying the pathogenesis of bacterially induced diseases.

ABSTRACTTo study what determines the arthritogenicity of bacterial cell walls, cell wall-induced arthritis in the rat was applied, using four strains of Lactobacillus. Three of the strains used proved to induce chronic arthritis in the rat; all were Lactobacillus casei. The cell wall of Lactobacillus fermentumdid not induce chronic arthritis. All arthritogenic bacterial cell walls had the same peptidoglycan structure, whereas that of L. fermentumwas different. Likewise, all arthritogenic cell walls were resistant to lysozyme degradation, whereas the L. fermentumcell wall was lysozyme sensitive. Muramic acid was observed in the liver, spleen, and lymph nodes in considerably larger amounts after injection of an arthritogenicL. caseicell wall than following injection of a nonarthritogenic L. fermentumcell wall. The L. caseicell wall also persisted in the tissues longer than theL. fermentumcell wall. The present results, taken together with those published previously, underline the possibility that the chemical structure of peptidoglycan is important in determining the arthritogenicity of the bacterial cell wall.

Yersinia enterocolitica serotype O:3 and O:8 urease-negative mutants unable to express the 19-kDa beta subunit of urease were constructed and tested for virulence and arthritogenicity. Our results indicate that urease is needed for full virulence in oral infections and that it is not an arthritogenic factor in the rat model.

ABSTRACTYersinia enterocoliticaserotype O:3 and O:8 urease-negative mutants unable to express the 19-kDa β subunit of urease were constructed and tested for virulence and arthritogenicity. Our results indicate that urease is needed for full virulence in oral infections and that it is not an arthritogenic factor in the rat model.

Outer membrane protein YadA, Yersinia adhesin, is one of the plasmid-encoded virulence factors of yersiniae. YadA protects bacteria against host defense through several different mechanisms. One important role of YadA is to mediate binding to several collagen types. Our recent study revealed that a yadA null mutant of Yersinia enterocolitica serotype O:8 has a drastically reduced arthritogenic capacity when injected intravenously into Lewis rats. To further characterize the arthritogenic role of YadA, we repeated the rat experiments with strain Y. enterocolitica O:8/pYV082; this strain expresses a YadA deletion derivative lacking 22 amino acids from the amino-terminal hydrophobic region and does not bind to collagen. Y. enterocolitica O:8/pYV082 induced arthritis in 5 to 14% of rats inoculated with arthritogenic doses, whereas the arthritis incidence with the wild-type parent strain was 65%. The parent strain was slightly more virulent than Y. enterocolitica O:8/pYV082, as determined by rat mortality. It also frequently induced skin abscesses, whereas Y. enterocolitica O:8/pYV082 did not. Infection kinetics in spleen and mesenteric lymph nodes were about the same with both of the bacterial strains used, and the same was true of the Yersinia-specific antibody response. Altogether, these results suggest that YadA-mediated collagen binding contributes to the arthritogenicity of Y. enterocolitica O:8.

The binding of live Yersinia enterocolitica to frozen sections of human intestine was investigated qualitatively by monitoring the binding of bacteria by using Gram or immunoperoxidase staining as well as quantitatively by a new enzyme immunoassay-on-slide method. We have demonstrated that the binding of various Y. enterocolitica serotypes and Escherichia coli clones to frozen sections of human intestine is mediated by the Yersinia adhesin, YadA. The YadA-mediated binding occurs mainly at the submucosal layer of the intestinal wall and only to a limited extent at the mucosal layer; there binding is mostly to the mucin threads. In addition, partially purified YadA binds to frozen sections with a pattern similar to that of intact bacteria. Collagen, laminin, or partially purified YadA only partially inhibited the YadA-mediated binding of bacteria, presumably because YadA is multifunctional. A combination of collagen and laminin inhibited the binding more efficiently. Therefore, YadA may be involved in the interactions with the extracellular matrix molecules after the invasion of the intestinal tissue.

The gene coding for a putative 19-kDa arthritogenic antigen of Yersinia enterocolitica O:3 (A. K. H. Mertz et al., J. Clin. Invest. 87:632-642, 1991) was cloned and sequenced after amplification of part of the gene by the polymerase chain reaction using degenerate primers, inferred from the amino acid sequence. The deduced amino acid sequence of the antigen showed similarity to small subunits of ureases from several different organisms, including the jack bean urease. Screening of a genomic library of Y. enterocolitica O:3 with a 19-kDa-antigen-specific DNA probe allowed recombinant clones containing the entire urease operon to be obtained. These clones expressed urease activity in Escherichia coli.

The rfb gene cluster of Yersinia enterocolitica O:3, responsible for the biosynthesis of the O side chain, was previously cloned, and a Y. enterocolitica O:3 side chain-specific bacteriophage (phi YeO3-12) was isolated (A. Al-Hendy, P. Toivanen, and M. Skurnik, Microb. Pathog. 10:47-59, 1991). This paper describes the isolation and characterization of the bacteriophage phi YeO3-12-resistant mutant of Y. enterocolitica O:3, YeO3-R2. Lipopolysaccharide isolated from YeO3-R2 lacked the O side chain, as evidenced by silver staining and by immunoblots probed with a Y. enterocolitica O:3 O side chain-specific monoclonal antibody. The core was complete, as shown in immunoblots probed with an outer core-specific monoclonal antibody. In Southern blotting with the cloned Y. enterocolitica O:3 rfb region as a probe, there was no detectable difference in the hybridization pattern of chromosomal DNA isolated from YeO3-R2 and that isolated from wild-type Y. enterocolitica O:3. This suggests that a point mutation, rather than a large deletion, was responsible for the rough phenotype of YeO3-R2. The virulence of YeO3-R2 was determined in an orally infected desferal-attenuated murine model. The mutant was approximately 50-fold less virulent than the isogenic wild type. The ability of YeO3-R2 to reexpress O side chain, and hence full virulence, was reconstituted by complementing the chromosomal mutation in trans with the distal 6.5 kb of the Y. enterocolitica O:3 rfb region. This same 6.5-kb fragment transcomplemented a transposon mutation in the same area of the Y. enterocolitica O:3 rfb region when expressed in Escherichia coli. This transcomplementation implies that the rfb region of Y. enterocolitica O:3 is organized into at least two separate operons.

Outer membrane protein YadA, the Yersinia adhesin, is one of the plasmid-encoded virulence factors of yersiniae. To evaluate the role of YadA in the pathogenesis of reactive arthritis experimentally, we used YadA- strain YeO8-116, a kanamycin GenBlock insertion mutant derived from Yersinia enterocolitica O:8 wild-type strain 8081. As control strains, a plasmid-cured derivative (8081-c) of 8081 and a YopH- mutant (8081-yoph) were used. In addition, YeO8-116, with the yadA mutation transcomplemented with plasmid pMW10, was used. YeO8-116 induced arthritis to a considerably lesser extent than did wild-type strain 8081 when inoculated intravenously into Lewis rats. In rats surviving for over 14 days after the bacterial inoculation, the arthritis incidences were 6% (4 of 72) among those inoculated with the yadA mutant and 51% (33 of 65) among those inoculated with wild-type strain 8081. When the yadA gene was transcomplemented back to YeO8-116, YeO8-116/pMW10 induced arthritis in 47% (9 of 19) of the inoculated rats. Plasmid-cured strain 8081-c did not induce arthritis in any of the 24 inoculated rats, whereas YopH- mutant 8081-yoph induced arthritis in 20% (5 of 25) of the rats inoculated. Although the 50% lethal dose of YeO8-116 was about sixfold higher than that of 8081, the kinetics of bacterial elimination from the spleen and mesenteric lymph nodes were about the same with both strains. Antibody responses in rats infected with the two strains were also indistinguishable. Our results indicate that YadA contributes to the arthritogenicity of Y. enterocolitica in the rat model.

Reactive arthritis is usually self-limiting polyarthritis, which develops after certain gastrointestinal or urogenital tract infections, mostly in susceptible HLA B27-positive individuals. In the pathogenesis of this arthritis, it is probably important that structures of the causative bacteria are found in the affected joints. The structure found in the synovial fluid phagocytes of the patients with reactive arthritis after Yersinia, Salmonella, and Shigella infections has always been lipopolysaccharide (LPS) of the causative bacteria. It has been in a highly processed form but still immunoreactive. To follow the degradation process of LPS, we fed peripheral blood monocytes of healthy blood donors with heat-killed Yersinia enterocolitica O:3 bacteria in vitro and monitored the fate of LPS by immunofluorescence and immunoblotting methods. Heat-killed bacteria were used since Y. enterocolitica O:3 bacteria are able to live inside monocytes in vitro and dividing intracellular bacteria would have made it impossible to monitor the degradation process of LPS with these methods. Both the core region and the O-polysaccharide chain of LPS persisted in cytoplasmic vacuoles and on plasma membrane of monocytes through the 7-day follow-up time. Migration properties of processed LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested structural modifications of LPS. We also demonstrated that core epitopes appearing on the surface of Yersinia-fed monocytes on day 4 of incubation were processed intracellularly, suggesting that LPS-containing phagocytes are a constant source of membrane-active LPS in their microenvironment as well as in the joints of arthritic patients.

We have determined the opsonic capacity of specific antibodies in patient sera obtained after Yersinia infection. The results indicate that Yersinia antibodies lead to complement activation through the classical pathway, thus overcoming the inhibition of complement-mediated opsonization in the absence of specific antibodies provided by the virulence plasmid in Yersinia enterocolitica and Yersinia pseudotuberculosis. Further, antibodies against plasmid-encoded structures, the Yersinia outer membrane proteins (YOPs), are not necessary in this effect. This is indicated by two facts. (i) Monoclonal antibodies directed against the O polysaccharide of Y. enterocolitica O:3 are capable of opsonizing the plasmid-containing bacteria through C1q binding. (ii) Rabbit antisera show opsonic activity when obtained by immunization both with plasmid-containing Y. enterocolitica expressing the YOPs and a plasmid-cured variant not expressing these proteins.