Tübingen, Univ., Diss., 2002.

Staphylococcus aureus (S. aureus) is a frequent cause of infections and sepsis in animals and humans worldwide. Staphylococcal enterotoxins and toxic shock syndrome toxin-1 are bacterial superantigens (SAgs) produced by S. aureus that simultaneously bind to T cell receptor (TCR) and the major histocompatibility complex (MHC) class II, leading to extensive T cell stimulation, release of cytokines, consequently resulting in toxic shock and immunosuppression. In this study, we generated mutant SAgs by introducing alanine substitution at residues involved in interaction with MHC class II and TCR binding and demonstrated attenuation of toxicity in vitro and in vivo. An immunization with mutant SAgs elicits production of neutralizing antibodies against wild type SAgs and protected animals from S. aureus peritonitis at a lethal dose. These results suggest that mutant SAgs will be useful to develop a novel vaccine against S. aureus infections.

Bovine mastitis is a significant disease affecting the dairy industry worldwide. The most frequently causative agent of contagious bovine mastitis is Staphylococcus aureus that produces numerous virulence factors contributing to its pathogenesis. However, it is not clearly defined which virulence factors play a critical role in bovine mastitis due to the heterogeneous virulence factor profiles in S. aureus isolated from different hosts and disease types. Among many virulence factors, it has long been postulated that staphylococcal cytotoxins and superantigens (SAgs) play an important role in the pathogenesis of S. aureus in bovine mastitis due to their potent toxicity toward host immune response. However, it has been a great challenge to determine the definite role of staphylococcal cytotoxins and SAgs in S. aureus pathogenesis due to the presence of multiple redundant cytotoxins and SAgs in a single S. aureus strain. Our longterm goal is to develop an effective vaccine to protect dairy cattle from S. aureus infection. The objective of this study is to; 1) determine the role of staphylococcal cytotoxins and superantigens in bovine mastitis; 2) develop a inducible and secretory expression vector and host system for a high production yield of recombinant protein; and 3) evaluate the protective effect of recombinant protein vaccine composed of staphylococcal cytotoxins and SAgs. The rationale of the proposed research is that development of an effective vaccine against S. aureus will prevent significant economic loss in the dairy industry and reduce the use of antibiotics in the dairy industry to prevent emergence of antibiotic resistance pathogens

Superantigens (SAgs) are implicated in the initiation of many diseases, including Kawasaki disease (KD), a multi-system vasculitis that leads to persistent inflammation and damage of coronary arteries. T cells are central to the pathogenesis of SAg-mediated diseases. Lactobacillus casei cell wall extract (LCWE) induces a disease in mice that resembles human KD, and contains a novel SAg. Despite the fact that SAg-activated T cells undergo apoptosis, they persist and are necessary for coronary inflammation in this model of KD. We report rescue from apoptosis of SAg-stimulated T cells in vitro by costimulation through CD28 or 4-1BB. CD28- or 4-1BB-mediated signaling stimulated concurrently with SAg upregulated the anti-apoptotic factor Bcl-XL. In vivo, co-injection of LCWE and a 4-1BB agonist aggravated coronary disease. These findings suggest that costimulation of T cells affects extent of illness in this model of KD, and supports targeting costimulation as a therapeutic intervention in SAg-triggered diseases.

Kawasaki disease (KD) is a multisystem vasculitis with predilection for the coronary arteries. Although the cause of KD remains elusive, there is evidence to suggest a superantigenic trigger. When T-cells are activated by a superantigen (SAg) they undergo massive proliferation but eventually apoptose; however, in KD, we hypothesize that these T-cells persist and infiltrate the coronary arteries. Previous studies have shown that enhanced costimulation through CD28 or 4-1BB rescues T-cells from apoptosis and exacerbates disease in a mouse model of KD. Our results suggest that this signal needs to be initiated close in timing to that of the SAg. In addition, the two molecules can act independently of one another, but are not additive. Also, stimulation of the 4-1BB pathway in the presence of a SAg elicits a Th1 phenotype. Lastly, TRAF1 regulates this enhanced survival downstream of 4-1BB. Thus, these results provide new insights into the effects of costimulation in SAg-mediated disease, and suggest that these pathways need to be targeted early to abrogate the enhanced survival of SAg-activated T-cells.

Superantigens (SAgs) are implicated in the initiation of many diseases, including Kawasaki disease (KD), a multi-system vasculitis that leads to persistent inflammation and damage of coronary arteries. T cells are central to the pathogenesis of SAg-mediated diseases. Lactobacillus casei cell wall extract (LCWE) induces a disease in mice that resembles human KD, and contains a novel SAg. Despite the fact that SAg-activated T cells undergo apoptosis, they persist and are necessary for coronary inflammation in this model of KD. We report rescue from apoptosis of SAg-stimulated T cells in vitro by costimulation through CD28 or 4-1BB. CD28- or 4-1BB-mediated signaling stimulated concurrently with SAg upregulated the anti-apoptotic factor Bcl-XL. In vivo, co-injection of LCWE and a 4-1BB agonist aggravated coronary disease. These findings suggest that costimulation of T cells affects extent of illness in this model of KD, and supports targeting costimulation as a therapeutic intervention in SAg-triggered diseases.

Kawasaki disease (KD) is a multisystem vasculitis with predilection for the coronary arteries. Although the cause of KD remains elusive, there is evidence to suggest a superantigenic trigger. When T-cells are activated by a superantigen (SAg) they undergo massive proliferation but eventually apoptose; however, in KD, we hypothesize that these T-cells persist and infiltrate the coronary arteries. Previous studies have shown that enhanced costimulation through CD28 or 4-1BB rescues T-cells from apoptosis and exacerbates disease in a mouse model of KD. Our results suggest that this signal needs to be initiated close in timing to that of the SAg. In addition, the two molecules can act independently of one another, but are not additive. Also, stimulation of the 4-1BB pathway in the presence of a SAg elicits a Th1 phenotype. Lastly, TRAF1 regulates this enhanced survival downstream of 4-1BB. Thus, these results provide new insights into the effects of costimulation in SAg-mediated disease, and suggest that these pathways need to be targeted early to abrogate the enhanced survival of SAg-activated T-cells.

Kawasaki disease (KD) is an acute inflammatory disease characterized by persistent inflammation of the coronary arteries. KD is characterized by the release of cytokines such as tumor necrosis factor alpha (TNFα) and is thought to be initiated by a superantigen (SAg). The Lactobacillus casei cell wall extract model of KD demonstrates a critical requirement for TNFα and its receptor during pathogenesis, although the precise effect of TNFα is unknown. A persistent T cell infiltrate in the coronary artery disagrees with established fates of SAg activated cells, which undergo apoptosis. In this work, TNFα was found to promote the survival of SAg-reactive T cells. The results demonstrate that TNFα regulates B7.2 molecule expression on antigen presenting cells, and that TNFα indirectly promotes the survival of SEB-stimulated T cells by driving costimulation. These observations demonstrate how TNFα prevents T cell apoptosis and lend support to KD therapies which target TNFα and B7.

Kawasaki disease (KD) is an acute inflammatory disease characterized by persistent inflammation of the coronary arteries. KD is characterized by the release of cytokines such as tumor necrosis factor alpha (TNFα) and is thought to be initiated by a superantigen (SAg). The Lactobacillus casei cell wall extract model of KD demonstrates a critical requirement for TNFα and its receptor during pathogenesis, although the precise effect of TNFα is unknown. A persistent T cell infiltrate in the coronary artery disagrees with established fates of SAg activated cells, which undergo apoptosis. In this work, TNFα was found to promote the survival of SAg-reactive T cells. The results demonstrate that TNFα regulates B7.2 molecule expression on antigen presenting cells, and that TNFα indirectly promotes the survival of SEB-stimulated T cells by driving costimulation. These observations demonstrate how TNFα prevents T cell apoptosis and lend support to KD therapies which target TNFα and B7.

Bacterial superantigens (SAgs) are virulence factors that induce nonspecific T-cell proliferation contributing to host immune avoidance, and occasionally severe life-threatening toxinoses such as toxic shock syndrome. In the current study, the multiple functions of 3 superantigens named staphylococcal enterotoxin-like toxins X, Y and Z are investigated. SElX and SElZ were non-emetic in a musk shrew model of emesis. SElX is structurally and phylogenetically related to staphylococcal superantigen-like proteins (SSls) which are non-mitogenic but exhibit a variety of immune modulatory properties. We carried out protein and gene expression analysis of mutants of different S. aureus gene regulators and demonstrated that selx expression is controlled by saeRS, a two-component regulator linked to the bacterial response to phagocytic signals. Considering the co-regulation of SElX with known mediators of innate immune evasion we investigated a potential role for SElX in both humoral and cellular innate immune modulation and discovered that SElX strongly binds to human, bovine, murine, and laprine neutrophils and interferes with IgG-mediated phagocytosis, independently of Fcγ receptor signalling. Bacterial survival assays with neutrophils demonstrated that the deletion of selx significantly reduced the ability of S. aureus to resist neutrophil killing. Site-directed mutagenesis in the conserved sialic acid-binding motif of SElX abolished its neutrophil binding capacity, which is consistent with a critical role for glycosylated receptors in this interaction. Importantly, the sialic-acid binding mutants of SElX retained the ability to induce T-cell proliferation demonstrating that the distinct functions of SElX are mechanistically independent. Affinity precipitation experiments identified potential glycoprotein receptors for SElX and the interaction with protein ICAM-3, an important ligand for MAC-1 integrins, was validated suggesting SElX may interfere with cell signalling. Taken together, we present the first example of a bi-functional SAg that can manipulate two distinct arms of the human immune system and contribute to S. aureus survival during infection.