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Biomedical subjects

Patrick M Schlievert

Publications and source records attributed to Patrick M Schlievert.

At least 37 records · Page 2Linked to original sources
PubMed2003

Functional analysis of the TCR binding domain of toxic shock syndrome toxin-1 predicts further diversity in MHC class II/superantigen/TCR ternary complexes.

Superantigens (SAGs) aberrantly alter immune system function through simultaneous interaction with lateral surfaces of MHC class II molecules on APCs and with particular variable regions of the TCR beta-chain (Vbeta). To further define the interface between the bacterial SAG toxic shock syndrome toxin-1 (TSST-1) and the TCR, we performed alanine scanning mutagenesis within the putative TCR binding region of TSST-1 along the central alpha helix adjacent to the N-terminal alpha helix and the beta7-beta9 loop as well as with two universally conserved SAG residues (Leu(137) and Tyr(144) in TSST-1). Mutants were analyzed for multiple functional activities, and various residues appeared to play minor or insignificant roles in the TCR interaction. The locations of six residues (Gly(16), Trp(116), Glu(132), His(135), Gln(136), and Gln(139)), each individually critical for functional activity as well as direct interaction with the human TCR Vbeta2.1-chain, indicate that the interface occurs in a novel region of the SAG molecule. Based on these data, a model of the MHC/TSST-1/TCR ternary complex predicts similarities seen with other characterized SAGs, although the CDR3 loop of Vbeta2.1 is probably involved in direct SAG-TCR molecular interactions, possibly contributing to the TCR Vbeta specificity of TSST-1.

Amino Acid Substitution↗
PubMed2003

Toxic shock syndrome in a horse with Staphylococcus aureus pneumonia.

A 3-year-old Thoroughbred gelding was examined because of clinical signs of pneumonia and shock. Mucous membrane petechiation and ventral edema were observed and considered to be a result of vasculitis. Epidermal necrosis developed on the distal portions of the limbs. The horse had a persistent high fever that was unresponsive to nonsteroidal anti-inflammatory treatment, and Staphylococcus aureus was isolated from a nasal swab specimen and 2 transtracheal wash fluid samples. Antimicrobial, anti-inflammatory, and supportive treatment resulted in clinical improvement. However, resolution of the pulmonary infection required long-term (42 days) antimicrobial administration. Staphylococcus aureus strains isolated from this horse were positive for the toxic shock syndrome toxin-1 gene and were shown to produce toxic shock syndrome toxin-1, the causative factor in toxic shock syndrome in humans. The horse's clinical signs were attributed to toxic shock syndrome secondary to pulmonary S. aureus infection.

Animals↗
PubMed2003

Penetration of toxic shock syndrome toxin-1 across porcine vaginal mucosa ex vivo: permeability characteristics, toxin distribution, and tissue damage.

OBJECTIVE: The purpose of this study was to evaluate transvaginal penetration of toxic shock syndrome toxin-1 and its effects on permeability and tissue integrity in vitro with the use of excised porcine vaginal mucosa. STUDY DESIGN: Permeability to tritiated water (1 and 10 microg/mL applied toxin) and transmucosal flux of (35)S-methionine-labeled toxic shock syndrome toxin-1 (10 and 20 microg/mL) for up to 12 hours were assessed with the use of a continuous flow perfusion system. The location of labeled toxin that penetrated the mucosal tissue strata was determined. The integrity of toxin-treated, intact, scalpel-incised tissue was evaluated histopathologically. RESULTS: Toxic shock syndrome toxin-1 caused a non-dose-dependent increase in mucosal permeability and traversed the intact mucosa at a low rate without disrupting tissue integrity. In incised vaginal mucosa, toxic shock syndrome toxin-1 induced subepithelial separation and atrophy that were analogous to clinically relevant vaginal lesions that were reported in fatal cases of menstrual toxic shock syndrome. CONCLUSION: An in vitro model could be used to demonstrate that toxic shock syndrome toxin-1 permeates the vaginal mucosa and distributes throughout the tissue. Histologic evaluation of tissues that were exposed to toxic shock syndrome toxin-1 demonstrated lesions that were similar to those lesions that were reported in cases of menstrual toxic shock syndrome.

Animals↗
PubMed2003

Structural, energetic, and functional analysis of a protein-protein interface at distinct stages of affinity maturation.

Due to a paucity of studies that synthesize structural, energetic, and functional analyses of a series of protein complexes representing distinct stages in an affinity maturation pathway, the biophysical basis for the molecular evolution of protein-protein interactions is poorly understood. Here, we combine crystal structures and binding-free energies of a series of variant superantigen (SAG)-major histocompatibility complex (MHC) class II complexes exhibiting increasingly higher affinity to reveal that this affinity maturation pathway is controlled largely by two biophysical factors: shape complementarity and buried hydrophobic surface. These factors, however, do not contribute equivalently to the affinity maturation of the interface, as the former dominates the early steps of the maturation process while the latter is responsible for improved binding in later steps. Functional assays reveal how affinity maturation of the SAG-MHC interface corresponds to T cell activation by SAGs.

Binding Sites↗
PubMed2003

The zinc-dependent major histocompatibility complex class II binding site of streptococcal pyrogenic exotoxin C is critical for maximal superantigen function and toxic activity.

The cocrystal structure of streptococcal pyrogenic exotoxin C (SPE C) with HLA-DR2a (DRA*0101,DRB5*0101) revealed a zinc-dependent interaction site through residues 167, 201, and 203 on SPE C and residue 81 on the beta-chain of HLA-DR2a (DRA*0101,DRB5*0101). Mutation of these SPE C residues resulted in dramatically reduced biological activities. Thus, the zinc-dependent major histocompatibility complex II binding site is critical for maximal biological function of SPE C.

Animals↗
PubMed2003

Genome diversification in Staphylococcus aureus: Molecular evolution of a highly variable chromosomal region encoding the Staphylococcal exotoxin-like family of proteins.

Recent genomic studies have revealed extensive variation in natural populations of many pathogenic bacteria. However, the evolutionary processes which contribute to much of this variation remain unclear. A previous whole-genome DNA microarray study identified variation at a large chromosomal region (RD13) of Staphylococcus aureus which encodes a family of proteins with homology to staphylococcal and streptococcal superantigens, designated staphylococcal exotoxin-like (SET) proteins. In the present study, RD13 was found in all 63 S. aureus isolates of divergent clonal, geographic, and disease origins but contained a high level of variation in gene content in different strains. A central variable region which contained from 6 to 10 different set genes, depending on the strain, was identified, and DNA sequence analysis suggests that horizontal gene transfer and recombination have contributed to the diversification of RD13. Phylogenetic analysis based on the RD13 DNA sequence of 18 strains suggested that loss of various set genes has occurred independently several times, in separate lineages of pathogenic S. aureus, providing a model to explain the molecular variation of RD13 in extant strains. In spite of multiple episodes of set deletion, analysis of the ratio of silent substitutions in set genes to amino acid replacements in their products suggests that purifying selection (selective constraint) is acting to maintain SET function. Further, concurrent transcription in vitro of six of the seven set genes in strain COL was detected, indicating that the expression of set genes has been maintained in contemporary strains, and Western immunoblot analysis indicated that multiple SET proteins are expressed during the course of human infections. Overall, we have shown that the chromosomal region RD13 has diversified extensively through episodes of gene deletion and recombination. The coexpression of many set genes and the production of multiple SET proteins during human infection suggests an important role in host-pathogen interactions.

Animals↗
PubMed2003

Characterization of Staphylococcus aureus enterotoxin L.

Staphylococcus aureus causes a wide variety of diseases. Major virulence factors of this organism include enterotoxins (SEs) that cause both food poisoning and toxic shock syndrome. Recently, a novel SE, tentatively designated SEL, was identified in a pathogenicity island from a bovine mastitis isolate. The toxin had a molecular weight of 26,000 and an isoelectric point of 8.5. Recombinant SEL shared many biological activities with SEs, including superantigenicity, pyrogenicity, enhancement of endotoxin shock, and lethality in rabbits when administered in subcutaneous miniosmotic pumps, but the protein lacked emetic activity. T cells bearing the T-cell receptor beta chain variable regions 5.1, 5.2, 6.7, 16, and 22 were significantly stimulated by recombinant SEL.

Animals↗
PubMed2003

Quorum sensing in Staphylococcus infections.

Quorum sensing via the accessory gene regulator (agr) system has been assigned a central role in the pathogenesis of staphylococci, particularly Staphylococcus aureus. While the control of virulence gene expression in vitro by agr has been relatively straightforward to describe, regulation of both the quorum response itself and virulence genes in vivo is considerably more complex. The quorum response is highly dependent upon the environment in which the organism is grown and is strongly influenced by additional regulators that respond to signals other than cell density. There is increasing evidence that the agr phenotype may influence the behavior and pathogenesis of biofilm-associated S. aureus and S. epidermidis and may contribute to the chronic nature of some biofilm-associated infections.

Bacterial Proteins↗
PubMed2002

Characterization of a novel staphylococcal enterotoxin-like superantigen, a member of the group V subfamily of pyrogenic toxins.

Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-like protein is directly 5' of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable region, beta chain (TCRVbeta) specific manner. T cells bearing TCRVbeta 2, 5.1, and 21.3 were significantly stimulated.

Amino Acid Sequence↗
PubMed2002

Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence.

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares approximately 1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A(2) (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

Amino Acid Sequence↗
PubMed2002

Formation of vegetations during infective endocarditis excludes binding of bacterial-specific host antibodies to Enterococcus faecalis.

Infectious endocarditis is a microbial infection of the endothelial lining of the heart that typically occurs on damaged or prosthetic heart valves. The characteristic lesion seen with infective endocarditis, termed "the vegetation," is composed of bacteria surrounded by a platelet/fibrin layer attached to the underlying endothelium. The vegetation has long been believed to exclude or hinder host defenses from clearing bacteria, although formal demonstration of mechanisms by which this occurs are lacking. This study investigated the ability of the vegetation to exclude host antibodies specific for the bacterial surface protein aggregation substance in vivo during experimental endocarditis caused by Enterococcus faecalis. The results demonstrate that, once the vegetation encloses bacteria, they are no longer accessible to high-titer bacterial-specific host antibodies, establishing a mechanism by which the vegetation functions to protect the bacteria from the humoral immune response.

Animals↗
PubMed2002

Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3. Implications for the evolution of staphylococcal pathogenicity islands.

We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island "modules."

Animals↗
PubMed2002

Structures of two streptococcal superantigens bound to TCR beta chains reveal diversity in the architecture of T cell signaling complexes.

Superantigens (SAGs) crosslink MHC class II and TCR molecules, resulting in an overstimulation of T cells associated with human disease. SAGs interact with several different surfaces on MHC molecules, necessitating the formation of multiple distinct MHC-SAG-TCR ternary signaling complexes. Variability in SAG-TCR binding modes could also contribute to the structural heterogeneity of SAG-dependent signaling complexes. We report crystal structures of the streptococcal SAGs SpeA and SpeC in complex with their corresponding TCR beta chain ligands that reveal distinct TCR binding modes. The SpeC-TCR beta chain complex structure, coupled with the recently determined SpeC-HLA-DR2a complex structure, provides a model for a novel T cell signaling complex that precludes direct TCR-MHC interactions. Thus, highly efficient T cell activation may be achieved through structurally diverse strategies of TCR ligation.

Animals↗
PubMed2002

Prevalence of superantigen-secreting bacteria in patients with Kawasaki disease.

OBJECTIVES: To assess the prevalence of superantigen secreting bacteria in children with acute Kawasaki disease (KD) relative to control patients. STUDY DESIGN: Bacterial cultures were obtained in a blinded fashion from the throat, rectum, and groin of 45 patients with untreated acute KD and 37 febrile control patients from 6 centers in the United States. Cultures were processed for the presence of superantigen-producing bacteria at a central laboratory. RESULTS: Staphylococci or streptococci that produced superantigens (TSST-1, SEB, SEC, SPEB, SPEC) were isolated from 25 of 45 patients with KD (56%) as compared with 13 of 37 (35%) control patients (P =.078). Because SEB- and SEC-producing Staphylococcus aureus have not been associated with KD and because they do not induce a Vbeta2+ T-lymphocyte response, we analyzed the difference between groups relative to superantigens TSST-1 or SPEB/SPEC production. TSST-1 secreting S aureus or SPEB/SPEC producing group A streptococci were isolated from 20 of 45 (44%) patients with KD compared with 7 of 37 (19%) control patients (P =.019). CONCLUSIONS: The overall isolation rates of superantigen (TSST-1, SPEB, SPEC, SEB, SEC) producing bacteria between patients with KD and febrile control patients were not statistically significant. However, future studies should further examine the potential role of Vbeta2-stimulatory superantigens (TSST-1 and SPEB/SPEC) in KD.

Antigens, Bacterial↗
PubMed2002

Characterization of two novel pyrogenic toxin superantigens made by an acute rheumatic fever clone of Streptococcus pyogenes associated with multiple disease outbreaks.

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vbeta1, Vbeta5.1, and Vbeta23, and SpeM had specificity for Vbeta1 and Vbeta23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.

Acute Disease↗