Toxoids of streptococcal pyrogenic exotoxin A are protective in rabbit models of streptococcal toxic shock syndrome.

Streptococcal pyrogenic exotoxins (SPEs) are superantigens that have been implicated in causing streptococcal toxic shock syndrome (STSS). Most notably, SPE serotype A is made by nearly all M-protein serotype 1 and 3 streptococci, the M types most associated with the illness (these strains contain one or more other SPEs, and those proteins are likely also to contribute to disease). We have prepared double-, triple-, and hexa-amino-acid mutants of SPE A by PCR and other mutagenesis procedures. The sites chosen for mutation were solvent-exposed residues thought to be important for T-cell receptor (TCR) or major histocompatibility complex (MHC) class II interaction. These mutants were nonsuperantigenic for human peripheral blood mononuclear cells and rabbit and mouse splenocytes and were nonlethal in two rabbit models of STSS. In addition, these mutants stimulated protective antibody responses. Interestingly, mutants that altered toxin binding to MHC class II were more immunogenic than mutants altering TCR binding. Collectively, these studies indicate that multiple-site mutants of SPE A are toxoids that may have use in protecting against the toxin's effects in STSS.

Aggregation and binding substances enhance pathogenicity in rabbit models of Enterococcus faecalis endocarditis.

We investigated the importance of enterococcal aggregation substance (AS) and enterococcal binding substance (EBS) in rabbit models of Enterococcus faecalis cardiac infections. First, American Dutch belted rabbits were injected intraventricularly with 10(8) CFU and observed for 2 days. No clinical signs of illness developed in animals given AS- EBS- organisms, and all survived. All rabbits given AS- EBS+ organisms developed signs of illness, including significant pericardial inflammation, but only one of six died. All animals given AS+ EBS- organisms developed signs of illness, including pericardial inflammation, and survived. All rabbits given AS+ EBS+ organisms developed signs of illness and died. None of the rabbits receiving AS+ EBS+ organisms showed gross pericardial inflammation. The lethality and lack of inflammation are consistent with the presence of a superantigen. Rabbit and human lymphocytes were highly stimulated in vitro by cell extracts, but not cell-free culture fluids, of AS+ EBS+ organisms. In contrast, cell extracts from AS- EBS- organisms weakly stimulated lymphocyte proliferation. Culture fluids from human lymphocytes stimulated with AS+/EBS+ enterococci contained high levels of gamma interferon and tumor necrosis factor alpha (TNF-alpha) and TNF-beta, which is consistent with functional stimulation of T-lymphocyte proliferation and macrophage activation. Subsequent experiments examined the abilities of the same strains to cause endocarditis in a catheterization model. New Zealand White rabbits underwent transaortic catheterization for 2 h, at which time catheters were removed and animals were injected with 2 x 10(9) CFU of test organisms. None of the animals given AS- EBS- organisms developed vegetations or showed autopsy evidence of tissue damage. Rabbits given AS- EBS+ or AS+ EBS- organisms developed small vegetations and had splenomegaly at autopsy. All rabbits given AS+ EBS+ organisms developed large vegetations and had splenomegaly and lung congestion at autopsy. Similar experiments that left catheters in place for 3 days revealed that all rabbits given AS- EBS- or AS+ EBS+ organisms developed vegetations, but animals given AS+ EBS+ organisms had larger vegetations and autopsy evidence of lung congestion. These experiments provide direct evidence that these two cell wall components play an important role in the pathogenesis of endocarditis as well as in conjugative plasmid transfer.

Analysis of toxicity of streptococcal pyrogenic exotoxin A mutants.

Streptococcal pyrogenic exotoxin A (SPE A) is secreted by some strains of Streptococcus pyogenes and is strongly associated with streptococcal toxic shock syndrome (STSS), a severe and often fatal illness. SPE A possesses a number of biological properties, some of which are shared with a group of exotoxins of streptococcal and staphylococcal origins, the pyrogenic toxin superantigens (PTSAgs). SPE A's most extensively studied property is superantigenicity. Superantigenic activation of T cells and monocytes stimulates the release of cytokines such as tumor necrosis factors alpha and beta, interleukin 1, and gamma interferon. These endogenous mediators are considered to be the primary cause of capillary leak, hypotension, and shock, the most severe manifestations of STSS. However, several studies have suggested that other properties of SPE A, such as ability to greatly enhance host susceptibility to endotoxin and ability to interact directly with endothelial cells, may play substantial roles in the syndrome. In this work we generated single- and double-site mutations of SPE A at residues K16, N20, C87, C90, C98, K157, S195, N20/C98, and N20/K157. The mutant SPE A's were analyzed in vivo for their lethal activity and in vitro for their superantigenic ability. Our results indicate that SPE A's ability to induce lethality and endotoxin enhancement does not require superantigenicity, and conversely superantigenicity does not necessarily lead to lethality. Thus, these properties and their relative contributions to the onset of hypotension and shock may be separable. Furthermore, evidence is presented that certain mutant toxins may be suitable for use as vaccine toxoids.

Acute group G streptococcal myositis associated with streptococcal toxic shock syndrome: case report and review.

The group G streptococcus (GGS) is a rare cause of deep soft-tissue infection. We report that we believe is the first case of acute diffuse GGS myositis in association with toxic shock. Although the causative organism did not contain the genes for group A streptococcal pyrogenic exotoxins (SPEs) or make SPEs, the organism produced at least one new toxin that shared the biologic properties of the SPEs.