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Biomedical subjects

P M Schlievert

Publications and source records attributed to P M Schlievert.

At least 19 recordsLinked to original sources
PubMed2004

A toroid model for in vitro investigations of toxic shock syndrome toxin-1 production.

Human behaviours and consumer products may affect vaginal microbial ecology, thereby influencing women's health. Relevant experimentation systems are needed to understand such possible links. Here, we describe the development of a practical semi-solid in vitro model to assess the effects of interactions between vaginal environment and the presence of tampons, on bacterial communities, including the production of toxic shock syndrome toxin-1 (TSST-1) by Staphylococcus aureus.

Bacterial Toxins↗
PubMed2003

Comparative molecular analysis of community- or hospital-acquired methicillin-resistant Staphylococcus aureus.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is a growing public health concern that has been associated with pediatric fatalities. It is hypothesized that the evolution of CA-MRSA is a recent event due to the acquisition of mec DNA by previously methicillin-susceptible strains that circulated in the community. This study investigated the genetic relatedness between CA-MRSA, hospital-associated MRSA (HA-MRSA), and nonmenstrual toxic shock syndrome (nmTSS) isolates. Thirty-one of 32 CA-MRSA isolates were highly related as determined by pulsed-field gel electrophoresis and spa typing yet were distinguishable from 32 HA-MRSA strains. The 31 related CA-MRSA isolates produced either staphylococcal enterotoxin B (n = 5) or C (n = 26), and none made TSS toxin 1. All CA-MRSA isolates tested contained a type IV staphylococcal cassette chromosome mec (SCCmec) element. In comparison, none of the HA-MRSA isolates (n = 32) expressed the three superantigens. Antibiotic susceptibility patterns were different between the CA-MRSA and HA-MRSA isolates; CA-MRSA was typically resistant only to beta-lactam antibiotics. Six of twenty-one nmTSS isolates were indistinguishable or highly related to the CA-MRSA isolates. MnCop, an nmTSS isolate obtained in Alabama in 1986, was highly related to the CA-MRSA isolates except that it did not contain an SCCmec element. These data suggest that CA-MRSA strains may represent a new acquisition of SCCmec DNA in a previously susceptible genetic background that was capable of causing nmTSS. CA-MRSA poses a serious health risk not only because it is resistant to the antibiotics of choice for community-acquired staphylococcal infections but also because of its ability to cause nmTSS via superantigen production.

Community-Acquired Infections↗
PubMed2001

Toxic shock syndrome after laminaria insertion.

BACKGROUND: Laminaria tents used to facilitate surgical abortion are rarely associated with significant infectious morbidity. CASE: A parous woman in midpregnancy had laminaria placed in her cervix followed by a second set after 24 hours. Eight hours later, she presented with dyspnea, hives, fever, tachycardia, and hypotension. Antibiotic treatment was initiated and a dilation and evacuation procedure was performed. Amniotic membrane cultures showed a heavy growth of Staphylococcus aureus with staphylococcal enterotoxin C expression, compatible with toxic shock syndrome. CONCLUSION: Laminaria cervical dilation might be associated with toxic shock syndrome.

Abortion, Induced↗
PubMed2001

Crystal structure of a superantigen bound to the high-affinity, zinc-dependent site on MHC class II.

MHC class II molecules possess two binding sites for bacterial superantigens (SAGs): a low-affinity site on the alpha chain and a high-affinity, zinc-dependent site on the beta chain. Only the former has been defined crystallographically. We report the structure of streptococcal pyrogenic exotoxin C (SPE-C) complexed with HLA-DR2a (DRA*0101, DRB5*0101) bearing a self-peptide from myelin basic protein (MBP). SPE-C binds the beta chain through a zinc bridge that links the SAG and class II molecules. Surprisingly, SPE-C also makes extensive contacts with the MBP peptide, such that peptide accounts for one third of the surface area of the MHC molecule buried in the complex, similar to TCR-peptide/MHC complexes. Thus, SPE-C may optimize T cell responses by mimicking the peptide dependence of conventional antigen presentation and recognition.

Animals↗
PubMed2001

Use of intravenous immunoglobulin in the treatment of staphylococcal and streptococcal toxic shock syndromes and related illnesses.

Pyrogenic toxin superantigens comprise a large family of exotoxins made by Staphylococcus aureus and group A streptococci. These toxins include toxic shock syndrome toxin-1, the staphylococcal enterotoxins, and the streptococcal pyrogenic exotoxins (synonyms: scarlet fever toxins and erythrogenic toxins), all of which have the ability to cause toxic shock syndromes and related illnesses. These toxins have a similar three-dimensional structure that allows them to interact with relatively invariant regions of major histocompatibility complex class II molecules on the surface of antigen-presenting cells and with certain variable regions of the T-cell receptor-beta chain. The consequence of these interactions (and other immunobiological properties of the toxins) is the exaggerated release of bioactive cytokines. The latter molecules are responsible for the clinical signs of illness associated with these toxins.

Dermatitis, Atopic↗
PubMed2001

Biochemical and biological properties of Staphylococcal enterotoxin K.

Staphylococcus aureus is an important human pathogen which is implicated in a wide variety of diseases. Major determinants of the virulence of this organism include extracellular virulence factors. Staphylococcal enterotoxins (SEs) are important causative agents in staphylococcal toxic shock syndrome and food poisoning. Our study identified a novel enterotoxin, SEK, and examined its biochemical and biological properties. SEK had a molecular weight of 26,000 and an experimentally determined pI of between 7.0 and 7.5. SEK was secreted by clinical isolates of S. aureus. We demonstrated that SEK had many of the biological activities associated with the SEs, including superantigenicity, pyrogenicity, the ability to enhance the lethal effect of endotoxin, and lethality in a rabbit model when administered by subcutaneous miniosmotic pump. Recombinant SEK was shown to stimulate human CD4(+) and CD8(+) T cells in a Vbeta-specific manner; T-cells bearing Vbeta 5.1, 5.2, and 6.7 were significantly stimulated to proliferate.

Amino Acid Sequence↗
PubMed2001

Comparative analysis of lipopolysaccharide-induced tumor necrosis factor alpha activity in serum and lethality in mice and rabbits pretreated with the staphylococcal superantigen toxic shock syndrome toxin 1.

Host susceptibility to lipopolysaccharide (LPS) is correlated with the levels of circulating tumor necrosis factor alpha (TNF-alpha) that develop in response to circulating LPS. Mice are resistant, relative to rabbits, to the lethal effects of LPS. This study indicates that mice and rabbits are equally sensitive to the lethal effects of circulating TNF-alpha but that mice are more resistant than rabbits to the induction of circulating TNF-alpha by LPS.

Animals↗
PubMed2001

Role of T cells and gamma interferon during induction of hypersensitivity to lipopolysaccharide by toxic shock syndrome toxin 1 in mice.

The superantigenic function of toxic shock syndrome toxin 1 (TSST-1) is generally regarded as an important determinant of its lethal effects in humans or experimental animals. This study examined the role of superantigenicity in a BALB/c mouse model of lethal TSST-1-induced hypersensitivity to lipopolysaccharide (LPS). In this model, TSST-1 greatly potentiated both LPS-induced lethality, as well as LPS-induced serum tumor necrosis factor alpha (TNF-alpha) activity. Although BALB/c-SCID mice were resistant to these LPS enhancement effects of TSST-1, BALB/c-SCID mice reconstituted with T cells were completely susceptible to the enhancement effect of TSST-1 on LPS-induced serum TNF-alpha. Mice pretreated with cyclosporine (Cs) or neutralizing antibodies against gamma interferon (IFN-gamma) did not develop lethal LPS hypersensitivity when injected with TSST-1, and these agents reduced the enhancement effect of TSST-1 on LPS-induced serum TNF-alpha by 99 and 85%, respectively. Cs pretreatment also completely inhibited the known capacity of TSST-1 to amplify LPS-induced levels of IFN-gamma in serum. In contrast, mice given Cs after a priming injection of TSST-1, but before LPS, still exhibited lethal hypersensitivity to LPS. Cs given after TSST-1 also did not inhibit enhancement of LPS-induced serum TNF-alpha by TSST-1 but inhibited the enhancement effect of TSST-1 on LPS-induced serum IFN-gamma by 50%. These experiments support the theory that TSST-1-induced hypersensitivity to LPS is mediated primarily by IFN-gamma derived from superantigen-activated T cells.

Adoptive Transfer↗
PubMed2001

Functional characterization of streptococcal pyrogenic exotoxin J, a novel superantigen.

Streptococcal toxic shock syndrome (STSS) is a highly lethal, acute-onset illness that is a subset of invasive streptococcal disease. The majority of clinical STSS cases have been associated with the pyrogenic toxin superantigens (PTSAgs) streptococcal pyrogenic exotoxin A or C (SPE A or C), although cases have been reported that are not associated with either of these exotoxins. Recent genome sequencing projects have revealed a number of open reading frames that potentially encode proteins with similarity to SPEs A and C and to other PTSAgs. Here, we describe the cloning, expression, purification, and functional characterization of a novel exotoxin termed streptococcal pyrogenic exotoxin J (SPE J). Purified recombinant SPE J (rSPE J) expressed from Escherichia coli stimulated the expansion of both rabbit splenocytes and human peripheral blood lymphocytes, preferentially expanded human T cells displaying Vbeta2, -3, -12, -14, and -17 on their T-cell receptors, and was active at concentrations as low as 5 x 10(-6) microg/ml. Furthermore, rSPE J induced fevers in rabbits and was lethal in two models of STSS. Biochemically, SPE J had a predicted molecular weight of 24,444 and an isoelectric point of 7.7 and lacked the ability to form the cystine loop structure characteristic of many PTSAgs. SPE J shared 19.6, 47.1, 38.8, 18.1, 19.6, and 24.4% identity with SPEs A, C, G, and H, streptococcal superantigen, and streptococcal mitogenic exotoxin Z-2, respectively, and was immunologically cross-reactive with SPE C. The characterization of a seventh functional streptococcal PTSAg raises important questions relating to the evolution of the streptococcal superantigens.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of a novel Staphylococcus aureus serine protease operon.

The present study identified and characterized a unique operon (spl) encoding six serine protease-like proteins. In addition, native Spl proteins were isolated and characterized. Typical of most exoproteins, the spl gene products contain putative 35- or 36-amino-acid signal peptides. The Spl proteins share 44 to 95% amino acid sequence identity with each other and 33 to 36% sequence identity with V8 protease. They also contain amino acids found in catalytic triads of enzymes in the trypsin-like serine protease family, and SplB and SplC were shown to degrade casein. The spl operon is transcribed on a 5.5-kb transcript, but several nonrandom degradation products of this transcript were also identified. Similar to other S. aureus exoprotein genes, the spl operon is maximally expressed during the transition into stationary phase and is positively controlled by the Agr virulence factor regulator. The Sar regulatory system did not affect spl operon expression. PCR analysis revealed the presence of the spl operon in 64% of the S. aureus isolates tested, although one spl operon-negative isolate was shown to contain at least two of the spl genes. Finally, intraperitoneal injection of an spl operon deletion mutant revealed no major differences in virulence compared to the parental strain.

Amino Acid Sequence↗
PubMed2001

Antibodies to a surface-exposed, N-terminal domain of aggregation substance are not protective in the rabbit model of Enterococcus faecalis infective endocarditis.

The aggregation substance (AS) surface protein from Enterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS(44-331)) was cloned with a C-terminal histidine tag translational fusion and expressed from Escherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS(44-331) reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS(44-331) bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS(44-331) were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS(44-331) were challenged intravenously with E. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due to E. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.

Amino Acid Sequence↗
PubMed2001

Identification of a novel two-component regulatory system that acts in global regulation of virulence factors of Staphylococcus aureus.

We have previously demonstrated that the presence of oxygen is necessary for the production of toxic shock syndrome toxin 1 (TSST-1) by Staphylococcus aureus in vitro. To investigate the mechanism by which oxygen might regulate toxin production, we identified homologs in S. aureus of the Bacillus subtilis resDE genes. The two-component regulatory system encoded by resDE, ResD-ResE, has been implicated in the global regulation of aerobic and anaerobic respiratory metabolism in B. subtilis. We have designated the S. aureus homologs srrAB (staphylococcal respiratory response). The effects of srrAB expression on expression of RNAIII (the effector molecule of the agr locus) and on production of TSST-1 (an exotoxin) and protein A (a surface-associated virulence factor) were investigated. Expression of RNAIII was inversely related to expression of srrAB. Disruption of srrB resulted in increased levels of RNAIII, while expression of srrAB in trans on a multicopy plasmid resulted in repression of RNAIII transcription, particularly in microaerobic conditions. Disruption of srrB resulted in decreased production of TSST-1 under microaerobic conditions and, to a lesser extent, under aerobic conditions as well. Overexpression of srrAB resulted in nearly complete repression of TSST-1 production in both microaerobic and aerobic conditions. Protein A production by the srrB mutant was upregulated in microaerobic conditions and decreased in aerobic conditions. Protein A production was restored to nearly wild-type levels by complementation of srrAB into the null mutant. These results indicate that the putative two-component system encoded by srrAB, SrrA-SrrB, acts in the global regulation of staphylococcal virulence factors, and may repress virulence factors under low-oxygen conditions. Furthermore, srrAB may provide a mechanistic link between respiratory metabolism, environmental signals, and regulation of virulence factors in S. aureus.

Aerobiosis↗
PubMed2001

Toxic shock syndrome and bacterial superantigens: an update.

Toxic shock syndrome (TSS) is an acute onset illness characterized by fever, rash formation, and hypotension that can lead to multiple organ failure and lethal shock, as well as desquamation in patients that recover. The disease is caused by bacterial superantigens (SAGs) secreted from Staphylococcus aureus and group A streptococci. SAGs bypass normal antigen presentation by binding to class II major histocompatibility complex molecules on antigen-presenting cells and to specific variable regions on the beta-chain of the T-cell antigen receptor. Through this interaction, SAGs activate T cells at orders of magnitude above antigen-specific activation, resulting in massive cytokine release that is believed to be responsible for the most severe features of TSS. This review focuses on clinical and epidemiological aspects of TSS, as well as important developments in the genetics, biochemistry, immunology, and structural biology of SAGs. From the evolutionary relationships between these important toxins, we propose that there are five distinct groups of SAGs.

Amino Acid Sequence↗
PubMed2000

Evidence for the involvement of bacterial superantigens in psoriasis, atopic dermatitis, and Kawasaki syndrome.

A growing body of evidence implicates streptococcal and staphylococcal superantigens in the development of psoriasis, atopic dermatitis and Kawasaki syndrome. In each of these illnesses, an abnormal state of immunologic activity is observed. Superantigens, which have a unique ability to activate large numbers of lymphocytes, are likely to contribute to these disorders in a number of ways. The demonstrated activities of bacterial superantigens include increasing the number of circulating lymphocytes, with activation of autoreactive subsets, upregulation of tissue homing receptors on circulating lymphocytes, and local activation of immune cells within affected tissues. Through these and other mechanisms, superantigens have a proven ability to induce high levels of inflammatory cytokines and/or initiate autoimmune responses that contribute to the development of skin and vascular disorders. Though development of the illnesses discussed in this review are highly complex processes, superantigens may well play a critical role in their onset or maintenance. Understanding superantigen function may elucidate potential therapeutic strategies for these disorders.

Dermatitis, Atopic↗
PubMed2000

Development of streptococcal pyrogenic exotoxin C vaccine toxoids that are protective in the rabbit model of toxic shock syndrome.

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.

Animals↗
PubMed2000

Mutational analysis of the superantigen staphylococcal exfoliative toxin A (ETA).

Exfoliative toxin A (ETA) is known to be a causative agent of staphylococcal scalded skin syndrome (SSSS). Although relatively little is known about exactly how the exfoliative toxins (ETs) cause SSSS, much has been discovered recently that may help elucidate the mechanism(s) by which ETA exhibits activities such as lymphocyte mitogenicity and epidermolytic activity. Here, we have shown that highly purified ETA does have T lymphocyte mitogenic activity in that wild-type ETA induced T cell proliferation whereas several single amino acid mutants lacked significant activity. Neither wild-type ETA nor any single amino acid mutants were proteolytic for a casein substrate, yet esterase activity was detected in wild-type ETA and several mutants, but eliminated in other mutants. A mutation in aa 164 (Asp to Ala) showed a 9-fold increase in esterase activity as well. Finally, we correlated esterase activity with epidermolytic activity. All mutants that lost esterase activity also lost epidermolytic activity. Conversely, mutants that retained esterase activity also retained exfoliative activity, implicating serine protease or serine protease-like activity in the causation of SSSS. Moreover, the mutants that displayed markedly reduced T cell superantigenic activity retained their epidermolytic activity (although some of these mutants required higher doses of toxin to cause disease), which suggests an ancillary role for this activity in SSSS causation.

Amino Acid Substitution↗
PubMed2000

Structural evidence for the evolution of pyrogenic toxin superantigens.

Pathogenic bacteria have evolved a wide variety of toxins to invade and attack host organisms. In particular, strains of the bacteria Staphylococcus aureus and Streptococcus pyogenes produce a family of pyrogenic toxin superantigens (PTSAgs) that can cause illness, e.g., toxic shock syndrome, or synergize with a number of other immune system disorders. The PTSAgs are all similar in size and have a conserved two-domain tertiary fold despite minimal amino acid sequence identity. The tertiary structure of PTSAg domain 1 is similar to the immunoglobulin binding motif of streptococcal proteins G and L. PTSAg domain 2 resembles members of the oligosaccharide/oligonucleotide binding fold family that includes the B subunits of the AB(5) heat-labile enterotoxins, cholera toxin, pertussis toxin, and verotoxin. The strong structural homology between the pyrogenic toxins and other bacterial proteins suggests that the PTSAgs evolved through the recombination of two smaller beta-strand motifs.

Amino Acid Motifs↗