Quantifying the temporal expression of the Staphylococcus aureus collagen adhesin.

The initial event in the pathogeneis of most Staphylococcus aureus infections is attachment to biological substrates in vivo using specific protein-protein interactions. We previously quantified the temporal expression of the collagen adhesin (CNA) on S. aureus Phillips and demonstrated that dynamic adhesin capacity to collagen is strongly influenced by the adhesin density. However, most strains of S. aureus express an extensive amount of Protein A on the surface, presenting a complication when using immunofluorescence to quantify specific surface adhesins. In this study, an improved adhesin quantification method is presented that accommodates variable surface Protein A levels. This method was used to examine the temporal expression of CNA on six S. aureus strains. Collagen adhesin levels varied with growth phase and strain, ranging from 1096 to 8950 copies per cell. Five of the six strains exhibited a temporal expression pattern similar to that previously reported for S. aureus Phillips.     

The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model.

We have studied the role of the accessory gene regulator (agr) of Staphylococcus aureus as a virulence determinant in the pathogenesis of septic arthritis. At least 15 genes coding for potential virulence factors in Staphylococcus aureus are regulated by a putative multicomponent signal transduction system encoded by the agr/hld locus. agr and hld mutants show a decreased synthesis of extracellular toxins and enzymes, such as alpha-, beta-, and delta-hemolysin, leucocidin, lipase, hyaluronate lyase, and proteases, and at the same time an increased synthesis of coagulase and protein A as compared with the wild-type counterpart. We have used a recently described murine model of S. aureus-induced arthritis to study the virulence of S. aureus 8325-4 and two agr/hld mutants derived from it. Sixty percent of the mice injected with the wild-type strain developed arthritis, whereas agrA and hld mutants displayed joint involvement in only 10 and 30%, respectively. In addition, 40% of the mice inoculated with the wild-type strain displayed an erosive arthropathy; such changes were not detectable at all in mice inoculated with the agrA mutant. Serum levels of interleukin-6, a potent B-cell differentiation factor, were significantly higher (P < 0.001) in the mice inoculated with the wild-type strain than in those inoculated with the agrA mutant counterpart. Overall, our results suggest that the agr system of S. aureus is an important virulence determinant in the induction and progression of septic arthritis in mice.     

Alpha-toxin and gamma-toxin jointly promote Staphylococcus aureus virulence in murine septic arthritis

Septic arthritis is a common and feared complication of staphylococcal infections. Staphylococcus aureus produces a number of potential virulence factors including certain adhesins and enterotoxins. In this study we have assessed the roles of cytolytic toxins in the development of septic arthritis by inoculating mice with S. aureus wild-type strain 8325-4 or isogenic mutants differing in the expression of alpha-, beta-, and gamma-toxin production patterns. Mice inoculated with either an alpha- or beta-toxin mutant showed degrees of inflammation, joint damage, and weight decrease similar to wild-type-inoculated mice. In contrast, mice inoculated with either double (alpha- and gamma-toxin-deficient)- or triple (alpha-, beta-, and gamma-toxin-deficient)-mutant S. aureus strains showed lower frequency and severity of arthritis, measured both clinically and histologically, than mice inoculated with the wild-type strain. We conclude that simultaneous production of alpha- and gamma-toxin is a virulence factor in S. aureus arthritis.     

Functional Analysis of the Staphylococcus aureus Collagen Adhesin B Domain

The Staphylococcus aureus collagen adhesin (CNA) occurs in at least four forms that differ in the number (one, two, three, or four) of B domains. The B domains contain 187 amino acids and are located between the domains that anchor CNA to the cell envelope and the ligand-binding A domain. To determine whether a B domain is required for functional expression of CNA, we cloned the 2B cna gene from S. aureus strain Phillips and then eliminated both B domains by overlapping PCR. The absence of a B domain did not affect processing of the collagen adhesin to the cell surface or the ability to bind collagen. Based on our recent demonstration that the capsule can mask CNA on the surface of S. aureus cells (A. F. Gillaspy et al., Infect. Immun. 66:3170-3178, 1998), we also investigated the possibility that multiple B domains can extend the ligand-binding A domain outward from the cell surface and thereby overcome the inhibitory effect of the capsule. Specifically, we cloned the naturally occurring 4B CNA variant from S. aureus UAMS-639 and, by successive elimination of B domains, generated 1, 2, and 3B variants that are isogenic with respect to the 4B clone. After introducing each variant into microencapsulated and heavily encapsulated strains of S. aureus and growing cells under conditions known to affect capsule production (e.g., growth on Columbia agar), we correlated capsule production with exposure of CNA on the cell surface and the ability to bind collagen. Under no circumstance was the masking effect of the capsule reduced by the presence of multiple B domains. These results indicate that the B domains do not extend the ligand-binding A domain outward in a fashion that can overcome the inhibition of collagen binding associated with capsule production.     

Presence and expression of collagen adhesin gene (cna) and slime production in Staphylococcus aureus strains from orthopaedic prosthesis infections.

Prosthesis-associated infections still represent one of the most serious complications in the clinical use of biomaterials. The most frequent causes are Staphylococcus aureus and Staphylococcus epidermidis. Several studies have been devoted to identify adhesion mechanisms for these bacteria. Slime in particular has been extensively investigated. Recently, in Staphylococcus aureus species, considerable attention has been given to the host protein receptors that have been shown in in vitro assays to serve as substrates for bacterial adhesion. Collagen-rich tissues, as bone and cartilage, that are the preferential sites of staphylococcal infections, are also the tissues that harbour orthopaedic implants. These can be easily coated in vivo by collagen and thus become prone to adhesion of Staphylococci strains which carry the collagen adhesin gene (cna). In this study the frequency of cna was determined within a collection of 35 Staphylococcus aureus strains from orthopaedic prosthesis infections by a PCR method. Also the collagen-binding ability and slime forming capacity was evaluated. 29% of the strains were cna-positive and also able to bind collagen in vitro. 83% of the strains were slime forming. The results indicate that in the examined bacterial population slime-positive strains predominate over the cna-positive strains, with a striking association of the two adhesion mechanisms in cna-positive strains.     

Preferential induction of septic arthritis and mortality by superantigen-producing staphylococci.

Staphylococcal enterotoxins A through D (SEA through SED) and toxic shock syndrome toxin-1 display superantigen properties, i.e., they stimulate a great fraction of T cells expressing certain T-cell receptor V beta sequences. Using a newly established rat model of septic Staphylococcus aureus arthritis, we have recently shown that an S. aureus strain producing SEA showed marked arthritogenic properties. In the present study we decided to employ another five S. aureus strains, each one producing a distinct exotoxin. Almost all rats injected with superantigen-producing strains developed arthritis. In contrast, only 20% of rats injected with an S. aureus strain lacking superantigen production displayed mild and transient arthritis. Mortality was preferentially induced among the rats inoculated with the S. aureus strains producing SEB and SED. This study emphasizes that superantigen production is an important virulence factor in the development of septic S. aureus arthritis. Differences concerning mortality between staphylococcal strains producing different exotoxins may be dependent on the degree of activation of the immune system.     

Ribonucleotide reductase class III, an essential enzyme for the anaerobic growth of Staphylococcus aureus, is a virulence determinant in septic arthritis

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.     

Genomic fingerprinting for epidemiological differentiation of Staphylococcus aureus clinical isolates.

We used genomic fingerprinting to investigate an outbreak of methicillin-resistant Staphylococcus aureus in the neonatal intensive care units (NICUs) of two hospitals. The hospitals are located in the same city and are part of the same medical care system. Fingerprinting was done by Southern blot hybridization with DNA probes for the genes encoding the S. aureus collagen adhesin (cna), fibronectin-binding proteins (fnbA and fnbB), and beta-toxin (hlb). Genomic DNA was digested with HaeIII (cna and fnbA-fnbB probes) or HindIII (hlb probe). Hybridization patterns could be distinguished on the basis of (i) the presence or absence of cna, (ii) the size of the restriction fragment containing the cna gene, (iii) restriction fragment length polymorphisms within fnbA and fnbB, (iv) the presence of a lysogenic phage within hlb, and (v) the sizes of the restriction fragments containing the phage-bacterial DNA junction fragments. Over a period of 4 months we examined a total of 46 isolates obtained from various wards within each hospital. Among these 46 isolates, we observed a total of 4 cna patterns, 11 fnbA-fnbB patterns, and 11 hlb patterns. Southern blots with HaeIII-digested genomic DNA and a combination of all three gene probes revealed a total of 16 clearly distinguishable patterns. A total of 22 of the 46 isolates were identical with respect to every genomic marker examined. A total of 21 of these 22 isolates were obtained from patients within an NICU. Nineteen of 21 isolates also exhibited identical antibiotic resistance profiles (antibiogram). Although 5 of the remaining 24 strains exhibited an antibiogram identical to those of the NICU isolates, all 24 strains could be distinguished from the NICU isolates by at least one genomic marker. These results suggest that the NICU isolates had a common origin and that genomic fingerprinting with the cna, fnbA, fnbB, and hlb gene probes can provide an important epidemiological tool for the identification of clinical isolates of S. aureus.     

The surface protein Pls of methicillin-resistant Staphylococcus aureus is a virulence factor in septic arthritis

Pls, a surface protein of certain methicillin-resistant Staphylococcus aureus strains, is associated with poor bacterial adherence to solid-phase fibronectin and immunoglobulin G, as well as with reduced invasion of cultured epithelial cells. Here the importance of Pls for the development of septic arthritis and sepsis was investigated by using a mouse model. Mice inoculated with a pls knockout mutant developed a much milder arthritis and showed less grave weight reduction than mice infected with the wild-type Pls(+) clinical isolate. Also, the pls mutant induced a significantly lower frequency of mortality than the wild-type strain. The bacterial load of the kidneys was larger in mice infected with the Pls(+) strain than in animals challenged with the pls mutant. However, there was no evident inflammatory effect due to the Pls molecule alone, as indicated by knee injection of purified Pls. In conclusion, the results show that Pls is a virulence factor for septic arthritis and sepsis.     

Borrelia burgdorferi RevA Significantly Affects Pathogenicity and Host Response in the Mouse Model of Lyme Disease

The Lyme disease spirochete, Borrelia burgdorferi, expresses RevA and numerous outer surface lipoproteins during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA is poised to interact with the extracellular matrix of the host. To further define the role(s) of RevA during mammalian infection, we created a mutant that is unable to produce RevA. The mutant was still infectious to mice, although it was significantly less well able to infect cardiac tissues. Complementation of the mutant with a wild-type revA gene restored heart infectivity to wild-type levels. Additionally, revA mutants led to increased evidence of arthritis, with increased fibrotic collagen deposition in tibiotarsal joints. The mutants also induced increased levels of the chemokine CCL2, a monocyte chemoattractant, in serum, and this increase was abolished in the complemented strain. Therefore, while revA is not absolutely essential for infection, deletion of revA had distinct effects on dissemination, arthritis severity, and host response.     

Overproduction of type 8 capsular polysaccharide augments Staphylococcus aureus virulence

Type 8 capsular polysaccharide (CP8) is the most prevalent capsule type in clinical isolates of Staphylococcus aureus. However, its role in virulence has not been clearly defined. CP8 strains such as strain Becker produce a small amount of capsule on their surface in vitro. In contrast, CP1 strains such as strain M produce a large amount of capsule, which has been shown to be an important antiphagocytic virulence factor. The cap8 and cap1 operons, required for the synthesis of CP8 and CP1, respectively, have been cloned and sequenced. To test whether CP8 contributes to the pathogenesis of S. aureus, we replaced the weak native promoter of the cap8 operon in strain Becker with the strong constitutive promoter of the cap1 operon of strain M. The resultant strain, CYL770, synthesized cap8-specific mRNA at a level about sevenfold higher than that in the parent strain. Remarkably, the CYL770 strain produced about 80-fold more CP8. In a mouse infection model of bacteremia, the CP8-overproducing strain persisted longer in the bloodstream, the liver, and the spleen in mice than the parent strain. In addition, strain CYL770 was more resistant to ospsonophagocytosis in vitro by human polymorphonuclear leukocytes. These results indicate that CP8 is an antiphagocytic virulence factor of S. aureus.     

Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis

Persistence of Staphylococcus aureus during invasive infections has been associated with a small-colony variant (SCV) phenotype. SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain. SCVs have been shown to be more resistant to antibiotics such as aminoglycosides, grow slowly and persist intracellularly. The aim of this study was to assess the virulence of an hemB mutant, which has been shown to display the typical characteristics of clinical SCVs, in a murine model of septic arthritis. NMRI mice were inoculated intravenously with either the wild type strain Newman or with its mutant displaying the SCV phenotype. The clinical, bacteriological, and histopathological progression of the disease was studied. Mice inoculated with the hemB mutant displayed a higher frequency and a significantly higher severity of arthritis than mice inoculated with the wild type Newman strain. Despite that, the mutant inoculated mice displayed significantly lower bacterial burden in their kidneys and joints compared with mice exposed to the wild parental strain. Notably, the hemB mutant produced almost 20 times more protease in vitro than the parental strain. We conclude that the small colony variants of S. aureus are more virulent on a per organism basis than its isogenic parental strain in the model of septic arthritis. This can at least in part be explained by the ability of SCV to produce high amounts of destructive proteases.     

The fate of temperature-sensitive salmonella mutants in vivo in naturally resistant and susceptible mice.

The in vivo net growth rate of salmonellae in mice is faster with virulent than with attenuated strains, and slower in resistant than in susceptible mice, the latter difference being controlled by a single host gene (Ity). Mice were injected intravenously (i.v.) with nonreplicating temperature-sensitive (TS) salmonellae mutants: TS mutants from virulent parents survived better in the RES than those from attenuated or non-virulent parents as if the latter were more susceptible to bactericidal mechanisms. However, a TS mutant from a virulent parent (Salmonella typhimurium C5) did not consistently survive better in susceptible C5) did not consistently survive better in susceptible Itys than in resistant Ityr mice, suggesting that this gene may not operate by a bactericidal mechanism. In many animals the TS salmonellae caused septic arthritis which first appeared at 2--3 weeks. Subcutaneous inoculation in the tail caused local lesions and the organism spread to the RES, but did not cause arthritis in the short term.     

The role of staphylococcal polysaccharide microcapsule expression in septicemia and septic arthritis.

Staphylococcus aureus arthritis is a rapidly progressive and highly erosive disease of the joints in which both host and bacterial factors are of pathogenic importance. One potential bacterial virulence factor is the ability to express a polysaccharide capsule (CP). Among 11 reported capsular serotypes, CP type 5 (CP5) and CP8 comprise 80 to 85% of all clinical blood isolates. The aim of this study was to assess the role of CP5 as a virulence factor in staphylococcal septicemia and septic arthritis with a recently established murine model of hematogenously spread S. aureus arthritis. NMRI mice were inoculated intravenously with S. aureus strains isogenic for expression of CP5, and clinical, bacteriological, serological, and histopathological progression of disease was studied. Inoculation of 7 x 10(6) CFU of S. aureus per mouse induced 55% mortality in the group inoculated with the CP-expressing bacteria, compared to 18% in the group inoculated with CP- mutants. A lower dose of inoculum (3 x 10[6] per mouse) did not give rise to mortality in mice inoculated with CP mutant strains, whereas 18% of the mice inoculated with the CP5-expressing S. aureus died. Importantly, mice inoculated with S. aureus expressing CP5 had a significantly higher frequency of arthritis and a more severe form of the disease. In vitro assays suggested that macrophages were not able to phagocytize CP5+ staphylococci as efficiently as they were CP5- strains. In addition, once phagocytized, CP5+ bacteria were less efficiently killed than CP- mutants. In summary, CP5 leads to a higher frequency of arthritis and a more severe course of the disease. This seems to be related to the effects of the downregulatory properties of CP on the ingestion and intracellular killing capacity of phagocytes. Our results clearly indicate that the expression of CP5 is a determinant of the virulence of S. aureus in arthritis and septicemia.     

Distribution of collagen adhesin gene among various types of Staphylococcus aureus strains associated with bovine mammary gland

A subset of 58 bovine mastitis-associated Staphylococcus aureus isolates with known coa types were investigated for the presence of collagen adhesin gene, cna. According to the enzyme restriction pattern of gene encoding coagulase, strains were divided into nine genotypes. All isolates were investigated by PCR for the presence of gene encoding Cna, which was considered as an important virulence factor associated with bacteria adhesion. Interestingly, 49 (84.5%) strains were found to be cna ⁺ with significant variations across the predominant and rare genotypes. According to the results of this study, it might be emphasized that cna is significantly more common in bovine mastitis-associated S. aureus isolates and distributed dependently between genotypes. The finding that collagen–adhesin is present in the majority of the bovine mastitis-associated S. aureus isolates encourages the development of new strategies to prevent mastitis, based on antagonist ligands able to interact with surface adhesin and block its specific binding with matrix collagen.     

Diversity of ace, a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of ace during human infections

Our previous work reported that most Enterococcus faecalis strains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46 degrees C, but not 37 degrees C, and we subsequently identified an E. faecalis sequence, ace, that encodes a bacterial adhesin similar to the collagen binding protein Cna of Staphylococcus aureus. In this study, we examined the diversity of E. faecalis-specific ace gene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine E. faecalis strains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5' region of ace that encodes the A domain was sequenced, and these sequences showed > or =97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of S. aureus, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived from E. faecalis strain OG1RF detected Ace in mutanolysin extracts of seven of nine E. faecalis strains after growth at 46 degrees C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients with E. faecalis endocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other E. faecalis antigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an E. faecalis endocarditis patient serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to collagen types I and IV and laminin. In conclusion, these results show that ace is highly conserved among isolates of E. faecalis, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.     

EmaA, a potential virulence determinant of Aggregatibacter actinomycetemcomitans in infective endocarditis

The gram-negative fastidious human oropharyngeal Aggregatibacter actinomycetemcomitans is implicated in the etiology of infective endocarditis. EmaA, an oligomeric coiled-coil adhesin homologous to YadA of Yersinia enterocolitica, was hypothesized to mediate the interaction of A. actinomycetemcomitans with collagen. Collagen, the most abundant protein in human bodies and the main component of extracellular matrix (ECM), predominates in the supporting tissue of cardiac valves. To extend our earlier studies using purified collagen to determine bacterial binding activities, we developed a tissue model using rabbit cardiac valves to investigate the interaction of A. actinomycetemcomitans with native collagen. The resected mitral valves, with or without removal of the endothelium, were incubated with equivalent numbers of the wild type and the isogenic emaA mutant defective in collagen binding. There was no difference in binding between the wild-type and the mutant strains when the endothelium remained intact. However, the emaA mutant was fivefold less effective than the wild-type strain in colonizing the exposed ECM. A 10-fold increase in the binding of the wild-type strain to ECM was observed compared with the intact endothelium. Similar observations were replicated in an in vivo endocarditis rabbit model; the emaA mutant was 10-fold less effective in the initial infection of the traumatized aortic valve. Colocalization studies indicated that A. actinomycetemcomitans bound to type I collagen. A. actinomycetemcomitans preferentially colonized the ECM and, together with the evidence that EmaA interacts with the native collagen, suggested that the adhesin is likely a potential virulence determinant of the bacterium in the initiation of infective endocarditis.     

Cable pili and the 22-kilodalton adhesin are required for Burkholderia cenocepacia binding to and transmigration across the squamous epithelium

Burkholderia cenocepacia strains expressing both cable (Cbl) pili and the 22-kDa adhesin bind to cytokeratin 13 (CK13) strongly and invade squamous epithelium efficiently. It has not been established, however, whether the gene encoding the adhesin is located in the cbl operon or what specific contribution the adhesin and Cbl pili lend to binding and transmigration or invasion capacity of B. cenocepacia. By immunoscreening an expression library of B. cenocepacia isolate BC7, we identified a large gene (adhA) that encodes the 22-kDa adhesin. Isogenic mutants lacking expression of either Cbl pili (cblA or cblS mutants) or the adhesin (adhA mutant) were constructed to assess the individual role of Cbl pili and the adhesin in mediating B. cenocepacia binding to and transmigration across squamous epithelium. Relative to the parent strain, mutants of Cbl pili showed reduced binding (50%) to isolated CK13, while the adhesin mutant showed almost no binding (0 to 8%). Mutants lacking either cable pili or the adhesin were compromised in their ability to bind to and transmigrate across the squamous epithelium compared to the wild-type strain, although this deficiency was most pronounced in the adhA mutant. These results indicate that both Cbl pili and the 22-kDa adhesin are necessary for the optimal binding to CK13 and transmigration properties of B. cenocepacia.     

Protein A is a virulence factor in Staphylococcus aureus arthritis and septic death

Staphylococcal protein A (SpA), a cell wall anchored protein of Staphylococcus aureus, has the ability to interact with several host components, possibly indicating a role as a virulence factor in S. aureus infections. In this study, the contribution of SpA to bacterial virulence was investigated in a murine model of S. aureus arthritis. Intravenous inoculation of S. aureus wild-type strain Newman gave rise to more severe arthritis and higher mortality than the isogenic spa mutant strain DU5873. The wild-type strain caused more in vitro spleen cell proliferation than the SpA-deficient strain. However, IL-6 and TNF-alpha levels were higher after stimulation with thespa mutant strain compared to the wild-type strain. To conclude, our results clearly indicate that SpA is a virulence factor of S. aureus in murine septic arthritis.     

Role of YadA in arthritogenicity of Yersinia enterocolitica serotype O:8: experimental studies with rats.

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.