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G A Bohach

Publications and source records attributed to G A Bohach.

At least 37 records · Page 2Linked to original sources
PubMed1998

A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II.

The three-dimensional structure of the complex between a T cell receptor (TCR) beta chain (mouse Vbeta8.2Jbeta2.1Cbeta1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR beta chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the beta-SEC3 complex ("hot spot" residues) are strictly conserved among enterotoxins reactive with mouse Vbeta8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vbeta-binding specificities.

Amino Acid Sequence↗
PubMed1998

Structural dichotomy of staphylococcal enterotoxin C superantigens leading to MHC class II-independent activation of T lymphocytes.

We have recently characterized an MHC class II-deficient human cell line, SW480, that supports the proliferation of purified human T cells in the presence of the staphylococcal enterotoxin and superantigen SEC1, but not the closely related isotypes SEC2 or SEC3. We now investigate the structural basis of this dichotomy and explore possible mechanisms that may account for it. Differences in activity between SEC1 and SEC2 were not attributable to differences in biochemical modification, to differences in Vbeta specificity, or to the potential to induce anergy. SEC2 inhibited SEC1-mediated T cell activation in the presence of SW480 cells, suggesting that SEC2 could compete with SEC1 for binding to the TCR but was unable to productively signal through the TCR. Utilizing a panel of hybrid enterotoxins we identified specific amino acids near the NH2-terminus of SEC1 that abrogated MHC class II-independent T cell activation, yet did not alter potency in the presence of class II+ APC. These residues mapped to the putative TCR binding domain of SEC1, and suggest that subtle differences in TCR binding affinity or the topology of the SEC1-TCR interaction can compensate for the lack of MHC class II and hence promote T cell proliferation.

Adenocarcinoma↗
PubMed1998

Intracellular Staphylococcus aureus escapes the endosome and induces apoptosis in epithelial cells.

We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes.

Animals↗
PubMed1998

Staphylococcus aureus Agr and Sar global regulators influence internalization and induction of apoptosis.

Staphylococcus aureus was recently shown to be internalized by and to induce apoptosis in a bovine mammary epithelial cell line, suggesting that these processes could be involved in staphylococcal pathogenesis or persistence. To examine the role of virulence factor regulators during internalization, mutant agr and sar strains of S. aureus were analyzed for their abilities to enter and induce apoptosis in epithelial cells. Like a previously characterized bovine mastitis isolate, the standard laboratory strain, RN6390 (wild type), entered the epithelial cells and subsequently induced apoptosis. In contrast, the mutant strains RN6911 (agr), ALC136 (sar), and ALC135 (agr sar) were internalized by the cultured cells at levels reproducibly greater than that for RN6390 but failed to induce apoptosis. The internalization of S. aureus was affected by growth phase, suggesting a role for agr-regulated surface proteins in this process. Furthermore, the ability to induce apoptosis required metabolically active intracellular bacteria. These data indicate that the ability of S. aureus to enter mammalian cells and induce apoptosis is dependent on factors regulated by Agr and Sar. Since transcriptional control by these global regulators is mediated by quorum-sensing and environmental factors, staphylococci may have the potential to induce several alternative effects on cells from an intracellular environment. A model for the function of the agr locus in the context of internalization, intracellular persistence, and dissemination is proposed.

Animals↗
PubMed1998

Characterization of staphylococcal bovine mastitis isolates using the polymerase chain reaction.

A polymerase chain reaction (PCR) assay was adapted to detect toxin genes of staphylococcal isolates from cases of bovine mastitis. Samples were obtained from three geographical areas: Korea and Idaho and Washington in the northwest United States. Samples from Korea and Washington were randomly chosen. Idaho samples were from a prospective study of mastitis etiology. Forty-one milk samples from 25 commercial farms in south-central Idaho were collected from cows with symptoms of mastitis. Although Staphylococcus aureus constituted 37.5% of mastitis isolates, these isolates lacked genes for staphylococcal enterotoxins (SEs), toxic shock syndrome toxin, and exfoliative toxins. In contrast, 4 of 13 isolates from Washington and 6 of 20 isolates from South Korea expressed SEs. These results suggest that PCR may be an effective means of screening bovine isolates for toxins. They also emphasize the potential for significant geographic differences in mastitis etiology.

Animals↗
PubMed1998

Induction of type 2 cytokines by a staphylococcal enterotoxin superantigen.

Persistent intramammary infections of dairy cows with Staphylococcus aureus may involve immunosuppression mediated by bacterial toxins such as enterotoxins and other super-antigens (SAgs). Previously we found that stimulation of bovine PBMC with staphylococcal enterotoxin C (SEC) induced a unique phenotype of activated CD8+ T cells expressing a newly identified activation molecule, ACT3. In the present study we found that SEC induced the expression of interleukin (IL)-4 and IL-10 mRNAs, two cytokines associated with type 2 responses. Elevated levels of IL-4 and IL-10, observed between day 0 and day 4 of culture, were associated with temporary inhibition of proliferative responses of T cells, evidenced by a decrease in numbers of CD4+ T cells and a small increase in numbers of CD8+ T cells. Vigorous proliferation of T cells occurred between days 4 and 7 of culture and with a bias towards CD8+ T cells. Acquisition of the ACT3+ phenotype by CD8+ T cells was preceded by induction of IL-4 mRNA. Thus, in the bovine system, SAgs may hinder protective responses by inducing type 2 cytokines, which interfere with immune clearance of many microbial pathogens. The results of the study are consistent with the hypothesis that SAgs are involved in immunosuppression, and suggest possible immunomodulatory mechanisms.

Animals↗
PubMed1997

A series of meso-tris (N-methyl-pyridiniumyl)-(4-alkylamidophenyl) porphyrins: synthesis, interaction with DNA and antibacterial activity.

A series of meso-5,10,15-tris(N-methyl-4-pyridiniumyl)-20-(4-alkylamidophen yl) porphyrins were synthesized by derivatizing the amino group on the phenyl ring with the following hydrophobic groups: -C(O)C7F15, -C(O)CH=CH2, C(O)CH3, -C(O)C7H15, and -C(O)C15H31. The cationic tris-pyridiumyl porphyrin core serves as a DNA binding motif and a photosensitizer to photomodify DNA molecules. The changes of the UV-Vis absorption spectra during the titration of these porphyrins with calf thymus DNA revealed a large bathochromic shift (up to 14 nm) and a hypochromicity (up to 55%) of the porphyrins Soret bands, usually considered as proof of porphyrin intercalation into DNA. Association constants (K) calculated according to the McGhee and von Hippel model, were in the range of 10(6)-10(7) M(-1). An increase in hydrophobicity of the substituents at the 20-meso-position produced higher binding affinity. These porphyrins caused photomodification of the supercoiled plasmid DNA when a green laser beam at 532 nm was applied. Those with higher surface activity acted more efficiently as DNA photomodifiers. The porphyrin with a perfluorinated alkyl chain (-COC7F15) at the meso-20-position inhibited the growth of gram-positive bacteria (S. aureus, or S. epidermidis). Other porphyrins exhibited moderate activity against both gram-negative and gram-positive organisms.

Anti-Bacterial Agents↗
PubMed1997

The structure of the superantigen exfoliative toxin A suggests a novel regulation as a serine protease.

Exfoliative toxin A (ETA) causes staphylococcal scalded skin syndrome which is characterized by a specific intraepidermal separation of layers of the skin. The mechanism by which ETA causes skin separation is unknown although protease or superantigen activity has been implicated. The X-ray crystal structure of ETA has been solved in two crystal forms to 2.1 and 2.3 A resolution and R-factors of 17% and 19%, respectively. The structures indicate that ETA belongs to the chymotrypsin-like family of serine proteases and cleaves substrates after acidic residues. The conformation of a loop adjacent to the catalytic site is suggested to be key in regulating the proteolytic activity of ETA through controlling whether the main chain carbonyl group of Pro192 occupies the oxyanion hole. A unique amino-terminal domain containing a 15-residue amphipathic alpha helix may also be involved in protease activation through binding a specific receptor. Substitution of the active site serine residue with cysteine abolishes the ability of ETA to produce the characteristic separation of epidermal layers but not its ability to induce T cell proliferation.

Amino Acid Sequence↗
PubMed1997

Purification of Staphylococcus aureus beta-toxin: comparison of three isoelectric focusing methods.

beta-Toxin (beta-hemolysin) is one of several extracellular proteins produced by Staphylococcus aureus. It is a sphingomyelinase which disrupts the membranes of erythrocytes and other mammalian cells. Despite its characterized mechanism of action, the role of beta-toxin in human and animal disease remains unclear. In this report, we compare three gentle, rapid methods to purify enzymatically active beta-toxin. Extracellular proteins in S. aureus strain RN4220 cell supernatants, containing a high concentration of the toxin, were precipitated by ethanol, dialyzed, and separated by preparative isoelectric focusing (IEF). We compared the efficiency of three preparative IEF methods: a Sephadex flat-bed IEF using pH 3.5-10.0 Ampholine, a liquid IEF using pH 7.8-8.9 Rotolyte buffers, and a liquid IEF with two consecutive steps using pH 3.0-10.0 Bio-Lytes in the first separation followed by a second step using pH 6.0-8.0 Bio-Lytes. All three IEF methods purified milligram amounts of enzymatically and biologically active beta-toxin. Typically, 2-5 mg of purified beta-toxin was obtained from 1.2 liters of culture medium. The total enzymatic activity recovered and overall yield were similar for all three methods. However, the single-step liquid IEF separation using Rotolyte buffers was the most preferred method because it purified beta-toxin to >95% purity, did not require dialysis to remove ampholytes, and was the most rapid of the three methods.

Bacterial Toxins↗
PubMed1997

Vbeta-dependent stimulation of bovine and human T cells by host-specific staphylococcal enterotoxins.

Staphylococcus aureus isolates from bovine and ovine species produce unique molecular variants of type C staphylococcal enterotoxin (SEC). The SEC animal variants have greater than 98% amino acid sequence identity with SEC1, a human-associated SEC. The two SEC animal variants have been designated SEC(bovine) and SEC(ovine) according to their corresponding host species. We showed previously that these toxins induce quantitatively different levels of T-cell stimulation in several animal species. The present study compared the abilities of these closely related host-specific SEC variants to stimulate Vbeta-bearing T cells from bovine and human donors. All three toxins expanded human T cells bearing T-cell receptor Vbeta elements (huVbeta) 3, 12, 13.2, 14, 15, 17, and 20. However, SEC1 resulted in greater expansion of hyVbeta12 than either SEC(bovine) or SEC(ovine). In addition, bovine T cells proliferate in a Vbeta-dependent manner in response to these superantigens (SAgs). All three toxins induced the proliferation of bovine T cells bearing the previously sequenced Vbeta element (boVbeta) from the bovine T-cell clone BTB13 (boVbetaBTB13). SEC1 and SEC(ovine) also were able to induce proliferation of bovine T cells bearing boVbetaBTB35, which SEC(bovine) failed to stimulate. The species-specific differences in T-cell proliferation exhibited by these closely related SEC variants may reflect the evolutionary adaptation of S. aureus, presumably to increase its host range by the manipulation of the immune system in a host-specific manner.

Amino Acid Sequence↗
PubMed1997

Characterization of the canine type C enterotoxin produced by Staphylococcus intermedius pyoderma isolates.

The type C staphylococcal enterotoxins (SECs) are a group of highly conserved proteins with substantial antigenic cross-reactivity. Although Staphylococcus intermedius and coagulase-positive species of staphylococci are reported to produce SEC and other SEs, toxins produced by species other than Staphylococcus aureus have not been previously characterized. In this study we report the molecular, biological, and immunological properties of the canine SEC (SECcanine) expressed by pathogenic isolates of S. intermedius. The mature form of SECcanine has 239 amino acid residues and a pI of 7.0. Typical of the SEs, purified SECcanine induces an emetic response in monkeys and the proliferation of T cells in a Vbeta-dependent manner. Although SECcanine has >95% sequence identity to previously described SEC variants, its sequence is most related to SEC2 and SEC3. In contrast to the sequence similarity, the Vbeta profile induced by SECcanine is typical of that induced by SEC1. This result is likely explained by the conservation of a cysteine residue at position 26 in SECcanine; residues at this position have been previously shown to determine subtype-dependent differences in T-cell receptor interactions of other SEs. Overall, these results show that superantigen toxins produced by the multiple members of the genus Staphylococcus are highly conserved in respect to biological and structural properties. Further, the frequent association of SECcanine with pyoderma in dogs supports the notion that the toxins are important for staphylococcal survival and pathogenesis.

Alleles↗
PubMed1996

Comparison of the beta-toxins from Staphylococcus aureus and Staphylococcus intermedius.

The beta-toxins produced by Staphylococcus aureus and Staphylococcus intermedius were purified to homogeneity from culture supernatants. Although the toxin from S. aureus has been throughly studied, less is known about its unique counterpart from S. intermedius. This is the first reported purification and analysis of the S. intermedius beta-toxin. Both toxins have similar enzymatic properties, belong to the class of neutral sphingomyelinases C, and have a high specificity for sphingomyelin. They also hydrolyze lysophosphatidylcholine at a much slower rate, but have no activity toward phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine. The kinetic parameters determined for both proteins (apparent Km 1.4 mM, Vmax 100 mmol/min/microg protein) are identical. Despite these similarities, the size and amino acid composition of the two beta-toxins differ. Molecular mass values, determined by electrophoresis and gel filtration, indicate that the both enzymes are single polypeptides. The decrease in sphingomyelinase activity of S. aureus beta-toxin upon pretreatment with dithiothreitol (DTT) indicates the presence of a disulfide bond in the protein. In contrast, DTT has no effect on the enzymatic activity of S. intermedius beta-toxin. This observation is consistent with the absence of detectable cysteine residue in the protein. N-terminal amino acid sequences determined for the first 19 residues of both beta-toxins also differ, only nine of the first 19 residues are identical. Further evidence that the two proteins differ was obtained by immunological analysis which demonstrated crossreactivity but a lack of identity.

Amino Acid Sequence↗
PubMed1996

Controlled release of antibiotics from coated orthopedic implants.

Chronic osteomyelitis is one of the most serious complications of orthopedic open fracture treatment. The objective of this study was to develop a biodegradable implant coating with impregnated antibiotics as an adjunct to current therapy. We used a polylactic-co-glycolic acid copolymer (PLGA) as the biodegradable carrier and gentamicin as the antibiotic. Our objectives were to establish elution characteristics of the antibiotic from the polymer, and determine if the coated orthopedic implants would inhibit bacterial growth in vitro. In the elution study, coated implants were incubated in phosphate buffered saline (PBS) at 37 degrees C and sampled daily for gentamicin levels. The in vitro model consisted of test tubes containing Mueller-Hinton culture broth inoculated with 5 x 10(6) cfu of Staphylococcus aureus and incubated at 37 degrees C. The implants were switched to a new set of inoculated tubes each day. Tubes were sampled for colony counting to determine bactericidal effects. Implant coatings consisted of 40 mg of gentamicin as a 20% mixture with PLGA. The elution curve showed an average level of 138 micrograms/mL over 15 days. This local concentration would be more than adequate to kill susceptible organisms. The in vitro study showed a significant reduction in bacterial growth in the test tubes containing coated implants. Control tubes averaged 2.5 x 10(8) cfu/mL of S.aureus over 24 days. Coated implant tubes averaged 0.9 cfu/mL. This was a reduction of greater than 99.999% (p < 0.0001). This study showed that a thin biodegradable implant coating can be developed with bactericidal activity against the organisms frequently associated with osteomyelitis in cases of open fractures.

Anti-Bacterial Agents↗
PubMed1996

Analysis of monoclonal antibodies reactive with molecules upregulated or expressed only on activated lymphocytes.

Monoclonal antibodies potentially specific for antigens expressed or upregulated on activated leukocytes were selected for further analysis from the panel submitted to the third international workshop on ruminant leukocyte antigens. The kinetics of expression of these activation antigens on resting peripheral mononuclear cells (PBMC) and PBMC stimulated with concanavalin A or staphylococcal superantigen SECI for 4, 24 or 96 h were compared, as well as their appearance on various subsets of cells. For some of them, a molecular mass could be determined after immunoprecipitation from radio-labeled, lectin-stimulated cells. Based on the results from the clustering, kinetic studies and biochemical data, evidence was gathered for assigning two additional mAbs to cluster BoCD25 (IL-2 receptor) and two mAbs to cluster BoCD71 (transferrin receptor). Four mAbs recognized an early activation antigen predominantly expressed on gamma delta T cells in short-term cultures. A number of other activation antigens were further characterized.

Animals↗
PubMed1996

Subtype-specific interactions of type C staphylococcal enterotoxins with the T-cell receptor.

The goal of this study was to investigate the molecular interaction between superantigens and the T-cell receptor (TCR). Using a quantitative polymerase chain reaction (PCR) to assess T-cell proliferation profiles, we found that SEB, SEC1, SEC2 and SEC3 expanded human T cells bearing V beta 3, V beta 12, V beta 13.2, V beta 14, V beta 15, V beta 17 and V beta 20. SEC2 and SEC3 have the additional ability to expand T cells bearing V beta 13.1, and their expansion of V beta 3 was markedly reduced compared to SEB and SEC1. Based on the activity of SEC1 mutants containing single amino acid substitutions, we concluded that the differential abilities of these native toxins to stimulate V beta 3 and V beta 13.1 was determined by the residue in position 26, located in the base of the SEC alpha 3 cavity. The SEC1 mutant, in which Val in position 26 was substituted with the analogous SEC2/SEC3 residue (Tyr), generated a V beta expansion profile that was indistinguishable from those generated by SEC2 and SEC3. Using these findings, the co-ordinates of a recently reported murine TCR beta-chain crystal structure, and other documented information, we propose a compatible molecular model for the interaction of SEC3 with the T-cell receptor. In this model complex, the complementarity-determining regions (CDRs) 1 and 2 and the hypervariable loop 4 of the V beta element contact SEC3 predominantly through residues in the alpha 3 cavity of the toxin. CDR3 of the beta chain is not involved in any toxin contacts. The proposed model not only includes contacts identified in previous mutagenesis studies, but is also consistent with the ability of tyrosine and valine in position 26 to differentially affect the expansion of V beta s 3 and 13.1 by the SEC superantigens.

CD3 Complex↗
PubMed1995

Molecular structure of staphylococcus and streptococcus superantigens.

Staphylococcus aureus and streptococci, notably those belonging to group A, make up a large family of true exotoxins referred to as pyrogenic toxin superantigens. These toxins cause toxic shock-like syndromes and have been implicated in several allergic and autoimmune diseases. Included within this group of proteins are the staphylococcal enterotoxins, designated serotypes A, B, Cn, D, E, and G; two forms of toxic shock syndrome toxin-1 also made by Staphylococcus aureus; the group A streptococcal pyrogenic exotoxins, serotypes A, B, and C; and recently described toxins associated with groups B, C, F, and G streptococci. The nucleotide sequences of the genes for all of the toxins except those from the groups B, C, F, and G streptococcal strains have been sequenced. The sequencing studies indicate that staphylococcal enterotoxins B and C and streptococcal pyrogenic exotoxin A share highly significant sequence similarity; staphylococcal enterotoxins A, D, and E share highly significant sequence similarity; and toxic shock syndrome toxin-1 and streptococcal pyrogenic exotoxin B and C share little, if any, sequence similarity with any of the toxins. Despite the dissimilarities seen in primary amino acid sequence among some members of the toxin family, it was hypothesized that there was likely to be significant three-dimensional structure similarity among all the toxins. The three-dimensional structures of three of the pyrogenic toxin superantigens have been determined recently. The structural features of two of these, toxic shock syndrome toxin-1 and enterotoxin C3, are presented. Toxic shock syndrome-1 exists as a protein with two major domains, referred to as A and B. The molecule begins with a short N-terminal alpha-helix that then leads into a clawshaped structure in domain B that is made up of beta strands.

Bacterial Toxins↗
PubMed1994

Persistent acylation of high-molecular-weight penicillin-binding proteins by penicillin induces the postantibiotic effect in Streptococcus pyogenes.

Penicillin at 10X MIC induced a postantibiotic effect (PAE) of 2.1 h in Streptococcus pyogenes. Progressive increases in the densities of penicillin-binding proteins (PBPs) 1-3 of the bacterium were detected at 30, 60, and 90 min during the postantibiotic phase. The increase in colony-forming units during this phase paralleled the kinetics of incorporation of lysine into proteins, suggesting that growth was triggered by de novo synthesis of PBPs. The question was raised as to whether the progressive increases in densities of PBPs were due to the restoration of preexisting PBPs or to synthesis of new PBPs. With 10X MIC of clindamycin to inhibit PBP synthesis during the postantibiotic phase, the temporal increase in densities of PBPs 1-3 were totally inhibited. These results suggest that the PAE of penicillin in S. pyogenes is caused by irreversible binding of penicillin to PBPs 1-3 and represents the time necessary for synthesis of new PBPs required for normal growth.

Acylation↗