Comparative inflammatory properties of staphylococcal superantigenic enterotoxins SEA and SEG: implications for septic shock

The severity of Staphylococcus aureus sepsis is positively associated with staphylococcal enterotoxin A (SEA) and negatively associated with the enterotoxin gene cluster (egc), which encodes five staphylococcal enterotoxins. We postulated that the variable, clinical severity of S. aureus sepsis might be a result of differences in the inflammatory properties of staphylococcal superantigens. We therefore compared the inflammatory properties of SEA with those of staphylococcal entérotoxin G (SEG), a member of the five egc superantigens. We found that SEA and SEG had similar superantigenic properties, as they induced CD69 expression on T lymphocytes and selective expansion of Vbeta subpopulations. Contrary to SEG, however, SEA induced a strong proinflammatory/Th1 response, including TNF-alpha and MIP-1alpha production. These results suggest that the association of SEA with the severity of S. aureus septic shock, characterized by a deleterious, inflammatory cascade, may be explained partly by the specific proinflammatory properties of this superantigen.     

The effect of site-specific monoclonal antibodies directed to toxic shock syndrome toxin-1 in experimental Staphylococcus aureus arthritis.

Staphylococcus aureus produces a large number of potential virulence factors, among these the superantigen toxic shock syndrome toxin-1 (TSST-1). We have recently demonstrated that TSST-1 is involved in the pathogenesis of septic arthritis. Recent data show that the TSST-1 molecule is composed of two distinct domains, one proposed to interact with T cell receptor (TCR) and one with the MHC class II. The aim of this study was to assess if interaction between TSST-1-specific MoAbs directed to sites on the MHC and/or TCR Vbeta affects the development of experimental S. aureus-induced arthritis. For that purpose we used a panel of seven MoAbs, which were injected intraperitoneally before and after inoculation with a TSST-1-producing S. aureus strain. Administration of antibodies did not affect the development of arthritis, suggesting inefficacy of such a procedure in neutralization of exotoxin-mediated disease manifestations.     

The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis.

The importance of a collagen-binding adhesin in the pathogenesis of septic arthritis has been examined by comparing the virulence of two sets of Staphylococcus aureus mutants in an animal model. Collagen adhesin-negative mutant PH100 was constructed by replacing the chromosomal collagen adhesin gene (cna) in a clinical strain, Phillips, with an inactivated copy of the gene. Collagen adhesin-positive mutant S. aureus CYL574 was generated by introducing the cna gene into CYL316, a strain that normally lacks the cna gene. Biochemical, immunological, and functional analyses of the generated mutants and their respective parent strains showed that binding of 125I-labeled collagen, expression of an immunoreactive collagen adhesin, and bacterial adherence to cartilage were directly correlated with the presence of a functional cna gene. Greater than 70% of the mice injected with the Cna+ strains developed clinical signs of arthritis, whereas less than 27% of the animals injected with Cna- strains showed symptoms of disease. Furthermore, mice injected with the Cna+ strain Phillips had remarkably elevated levels of immunoglobulin G1 and interleukin-6 compared with mice injected with the Cna- mutant PH100. Taken together, these results demonstrate that collagen adhesin plays an important role in the pathogenesis of septic arthritis induced by S. aureus.     

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.     

Repeated treatment with S. aureus superantigens expands the survival rate of Ehrlich ascites tumor bearing mice

The bacterial superantigen Staphylococcal enterotoxin-A (SEA), produced by some strains of Staphylococcus aureus, causes proliferation of cytotoxic T-lymphocytes and cytokine production in vivo. SEA has been shown to be highly efficient for antibody-targeted superantegen immunotherapy for different tumor models. A candidate B-cell superantigen that has received considerable attention these days is staphylococcal protein-A (PA). It has been shown to possess multiple immunological responses. The anti-tumor property of PA is well documented in the literature in various transplantable tumors of rats and mice. In the present study, we have shown that the T-cell superantigen SEA and B-cell superantigen PA induce immunomodulatory and anti-tumor activity which is strongly protentiated by PA + SEA co-administration. Combination treatment with PA and SEA prolongs the immune response in vivo, limits the development of immunological unresponsiveness and promotes maximum anti-tumor effects to tumor carrying animals, as compared with PA or SEA alone. The immune response after combined therapy is characterized by substantially augmented IFN-gamma, TNF-alpha, Nitric oxide and strong CTL activity. Our data demonstrate that combined PA + SEA therapy induces long-term survival of the animals, carrying the Ehrlich ascites tumor.     

Evidence for a disease-promoting effect of Staphylococcus aureus-derived exotoxins in atopic dermatitis

BACKGROUND: The skin of patients with atopic dermatitis (AD) exhibits a striking susceptibility to colonization with Staphylococcus aureus. Some strains of S aureus secrete exotoxins with T-cell superantigen activity (toxigenic strains), and abnormal T-cell functions are known to play a critical role in AD. OBJECTIVE: Our purpose was to examine the impact of superantigen production by skin-colonizing S aureus on disease severity. METHODS: In a cross-sectional study of 74 children with AD, the presence and density of toxigenic and nontoxigenic strains of S aureus was correlated with disease severity. In a subgroup of patients the T-cell receptor Vbeta repertoire of peripheral blood and lesional T cells was investigated and correlated with individual superantigen activity of skin-colonizing S aureus. RESULTS: Fifty-three percent of children with AD were colonized with toxigenic strains of S aureus producing staphylococcal enterotoxin C, staphylococcal enterotoxin A, toxic shock syndrome toxin-1, staphylococcal enterotoxin B, and staphylococcal enterotoxin D in decreasing frequency. Children colonized with toxigenic S aureus strains had higher disease severity compared with the nontoxigenic and S aureus-negative groups. Patients colonized with toxigenic S aureus exhibited shifts in the intradermal T-cell receptor Vbeta repertoire that correspond to the respective superantigen-responsive T-cell subsets. CONCLUSION: The data demonstrate that S aureus-released exotoxins can modulate disease severity and dermal T-cell infiltration.     

Are B lymphocytes of importance in severe Staphylococcus aureus infections?

To investigate the role of B cells in experimental, superantigen-mediated Staphylococcus aureus arthritis and sepsis, we used gene-targeted B-cell-deficient mice. The mice were inoculated intravenously with a toxic shock syndrome toxin 1 (TSST-1)-producing S. aureus strain. The B-cell-deficient and thus agamma-globulinemic mice showed striking similarities to the wild-type control animals with respect to the development of arthritis, the mortality rate, and the rate of bacterial clearance. Surprisingly, we found that the levels of gamma interferon in serum were significantly lower (P < 0. 0001) in B-cell-deficient mice than in the controls, possibly due to impaired superantigen presentation and a diminished expression of costimulatory molecules. In contrast, the levels of interleukin-4 (IL-4), IL-6, and IL-10 in serum were equal in both groups. Our findings demonstrate that neither mature B cells nor their products significantly contribute to the course of S. aureus-induced septic arthritis.     

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.     

Mucosal tolerance to a bacterial superantigen indicates a novel pathway to prevent toxic shock

Enterotoxins with superantigenic properties secreted during systemic Staphylococcus aureus infection are responsible for toxic shock. We show that intranasal administration of staphylococcal enterotoxin A (SEA), but not a recombinant SEA lacking superantigenic activity, protected mice against lethal systemic SEA challenge. Protection was superantigen specific since intranasal exposure to SEA would not protect against death caused by subsequent toxic shock syndrome toxin 1 systemic challenge. Protection was neither due to selective depletion of SEA-specific T-cell receptor Vbeta families nor due to production of neutralizing anti-SEA antibodies. Importantly, the production of interleukin 10 (IL-10) induced by "tolerization" (that is, by the induction of immunological tolerance) contributed to the observed protection against lethal superantigen-triggered disease. In support of this notion we found that (i) significantly increased levels of IL-10 in sera of "tolerized" animals (that is, animals rendered tolerant) and (ii) IL-10(-/-) mice could not be tolerized by mucosal SEA administration. Altogether, this is the first study to show that mucosal tolerance to a superantigen is readily triggered by means of immunodeviation.     

REPEATED TREATMENT WITH S. AUREUS SUPERANTIGENS EXPANDS THE SURVIVAL RATE OF EHRLICH ASCITES TUMOR BEARING MICE

The bacterial superantigen Staphylococcal enterotoxin-A (SEA), produced by some strains of Staphylococcus aureus, causes proliferation of cytotoxic T-lymphocytes and cytokine production in vivo. SEA has been shown to be highly efficient for antibody-targeted superantegen immunotherapy for different tumor models. A candidate B-cell superantigen that has received considerable attention these days is staphylococcal protein-A (PA). It has been shown to possess multiple immunological responses. The anti-tumor property of PA is well documented in the literature in various transplantable tumors of rats and mice. In the present study, we have shown that the T-cell superantigen SEA and B-cell superantigen PA induce immunomodulatory and anti-tumor activity which is strongly protentiated by PA+SEA co-administration. Combination treatment with PA and SEA prolongs the immune response in vivo, limits the development of immunological unresponsiveness and promotes maximum anti-tumor effects to tumor carrying animals, as compared with PA or SEA alone. The immune response after combined therapy is characterized by substantially augmented IFN-γ, TNF-α, Nitric oxide and strong CTL activity. Our data demonstrate that combined PA+SEA therapy induces long-term survival of the animals, carrying the Ehrlich ascites tumor.     

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.     

Experimental Arthritis in the Rat Induced by the Superantigen Staphylococcal Enterotoxin A

The pathogenesis of rheumatoid arthritis (RA) is incompletely understood. Human endogenous retroviruses (HERVs) and their superantigenic envelope protein (env) have been implicated in the pathogenesis of RA. In the present investigation, the arthritogenic potential of the superantigen staphylococcal enterotoxin A (SEA) has been investigated. In the present investigation, the bacterial superantigen staphylococcal enterotoxin A (SEA) was injected into the right knee joint of 15 Lewis rats. Further nine animals received saline. Animals were sacrificed one, five and 10 days after the injection, respectively. The antigens CD3, CD4, CD8, MHC class I, MHC class II, Pax5 and CD138 were investigated by immunohistochemistry on cryo‐sections. After intra‐articular SEA injection, the inflammation was initially dominated by CD8+ T cells. In the course of the investigation, the numbers of CD4+, Pax5+, CD138+ and MHC class II+ cells increased. CD3 was expressed in low numbers as compared to CD8. After saline injection, no similar inflammatory response has been detected. The arthritis induced by the superantigen SEA may be a novel model for inflammatory joint diseases, that is rheumatoid arthritis or juvenile idiopathic arthritis.     

Synergistic immunopotentiating effects induced by T-cell and B-cell superantigen in mice

Staphylococcal Enterotoxin-A(SEA), a 27kDa monomeric protein, produced by some strains of Staphylococcus aureus, is a prototype T-cell superantigen which causes proliferation of cytotoxic T-lymphocytes and produces cytokines like TNF-alpha and IFN-gamma. Recently Protein A (PA), a 42 kDa membrane protein of the Staphylococcus aureus Cowan-I strain, has been termed a B-cell super antigen. It has been shown to cause multiple immunological responses. In the present study we examined the effect of these two superantigens used separately as well as combination in a normal mouse system. It has been shown that combination treatment of PA and SEA is more effective than that of each individual one. FACS analyses of cell cycles showed that a finely turned cellular collaboration occurred in various phases of cell growth and proliferative response compared with controls (P<0.01). It has also been shown that the percentage of various cell types bearing different clusters of differentiation markers, e.g., CD8+, CD34+ increases considerably due to the combined effect of PA and SEA. We also observed that co-administration of both the elicits different soluble mediators like cytokines (TNF-alpha, INF-gamma, IL-1beta). No apoptotic phenomenon was observed (from the cell cycle analysis) for the dose of PA and SEA, used for the experiments, suggesting that these doses of PA and SEA should be non-toxic.     

Detection of Staphylococcus aureus enterotoxins A to D by real-time fluorescence PCR assay

Staphylococcus aureus is one of the most significant pathogens causing nosocomial and community-acquired infections. Among the secreted staphylococcal virulence factors, there is a growing list of enterotoxins which can induce gastroenteric syndrome and toxic shock syndrome. Here, we developed a real-time fluorescence PCR assay (TaqMan PCR) for the detection of genes encoding staphylococcal enterotoxins A, B, C1, and D (SEA, SEB, SEC1, and SED) of S. aureus as well as the mecA gene encoding methicillin resistance and the femB gene as a specific genomic marker for S. aureus. SEA to SED were selected because they are the four classically described enterotoxins of S. aureus and because they were detected by latex agglutination. In order to evaluate the reliability of TaqMan PCR, we investigated 93 isolates of S. aureus derived from patients at our hospital over 5 months and compared the results with data obtained by a commercially available reversed passive latex agglutination assay (SET-RPLA) for these isolates. Thirteen enterotoxin genes were detected by TaqMan PCR; however, no proteins expressed by these genes were detected by SET-RPLA. As a result, more isolates of S. aureus (n = 44) were found positive by TaqMan PCR for one or more enterotoxin genes than by SET-RPLA for the respective proteins expressed by these genes (n = 40). We conclude that TaqMan PCR is more sensitive because it offers the possibility for determining enterotoxins on a genotypic basis. Additionally, the assay allows the parallel detection of genes for SEA to SED and methicillin resistance in S. aureus. Furthermore, real-time PCR is well suited for screening large numbers of samples at the same time, allowing rapid, reliable, efficient, and cost-saving routine laboratory diagnosis.     

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.     

Experimental Staphylococcus aureus arthritis in mice.

Staphylococcus aureus arthritis is usually caused by bacteremia and is highly destructive. Controlled studies on septic arthritis in humans are difficult to perform, because the time of onset of the infection is unknown. Animal models of bacterial arthritis make it possible to control important variables in experimental studies. We present a mouse model of S. aureus arthritis in which the intravenous administration of 10(7) cells of S. aureus LS-1 induced arthritis or osteitis or both within 3 weeks in 80 to 90% of the mice. Signs of arthritis emerged within the first few days after the injection. An interesting finding was that the S. aureus strain used in this study binds bone sialoprotein, a glycoprotein known to be specifically localized to bone tissue. This new model of S. aureus arthritis enables the study of the kinetics of joint destruction and the host-bacterium relationship as well as therapeutical approaches to septic arthritis and osteomyelitis.     

First report of septicemia caused by an obligately anaerobic Staphylococcus aureus infection in a human

In this case report, we describe the first instance of septicemia caused by an obligately anaerobic Staphylococcus aureus in a human. A 45-year-old man presented with septicemia, septic arthritis, and multiple pulmonary abscesses, which were caused by an obligately anaerobic S. aureus. The clinical and microbiological features that led to the diagnosis are discussed. Genotyping cannot at present reliably separate S. aureus subsp. aureus from S. aureus subsp. anaerobius, but phenotypic characteristics suggest that the present isolate is a previously undescribed strain of anaerobic Staphylococcus aureus.     

Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria

Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains of S. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.     

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.