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.

Development of monoclonal antibodies to detect bovine FOXP3 in PBMCs exposed to a staphylococcal superantigen.

The role of regulatory T cells (Tregs) is well documented in immune homeostasis and protection against autoimmune disease. Forkhead box protein 3 (FOXP3) has been shown to be essential for the development and function of T(reg). Due to the lack of tools for FOXP3 detection in certain species, understanding the role of Treg in a variety of ruminant diseases has been hampered. In this study, we developed monoclonal antibodies (mAbs) against bovine FOXP3 using recombinant bovine FOXP3 lacking the forkhead domain as an immunogen. The specificity of the mAbs was confirmed by immunoblot and mass spectrometry. Expression of FOXP3 was induced in bovine PBMCs after 6 d of exposure to staphylococcal enterotoxin type C1 (SEC1) in vitro. Similar to findings in mice and humans, expression of FOXP3 was restricted to CD4+ CD25+ T cells. Transcriptional analysis of bovine TCR variable regions of the beta chain (boVbeta) showed that transcription of boVbeta sequences reactive with SEC1 increased for 6 d, and then boVbeta sequences non-reactive with SEC1 rapidly increased in the cultures. This indicates that induction of FOXP3+ CD4+ CD25+ Tregs by SEC1 is not Vbeta restricted. The FOXP3 mAbs developed in this study will be useful in the further investigation of the role of Treg in staphylococcal pathogenesis in bovine mastitis and other ruminant diseases.

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.

Application of extended single-reaction multiplex polymerase chain reaction for toxin typing of Staphylococcus aureus isolates in South Korea.

The extended single-reaction multiplex PCR (esr-mPCR) developed in this study to detect staphylococcal enterotoxins (SEs), including SEA, SEB, SEC, SED, SEE, SEH, SEI, and SEJ, requires fewer sets of primers than other conventional multiplex PCRs and can be used to detect newly identified staphylococcal enterotoxins SEs more readily. Esr-mPCR analysis of 141 isolates of Staphylococcus aureus obtained from abattoir and livestock product samples revealed that 27 of the S. aureus isolates were toxigenic, and two were 2 multitoxigenic isolates. The most prevalent SE type was SEI followed by SEA and SEH. In addition, we investigated the clonal relatedness of toxigenic S. aureus isolates by arbitrarily primed PCR (AP-PCR). AP-PCR analysis of toxigenic S. aureus isolates revealed that the discriminatory power of AP-PCR was 9 (D=0.81), 8 (D=0.77), and 10 types (D=0.83) with primers AP1, ERIC2, and AP7, respectively. The combination of three each AP-PCR result could rearrange toxigenic S. aureus isolates into 10 types and five subtypes, with the D-value of 0.92. Interestingly, our data showed that toxigenic S. aureus isolates from different sources had different fingerprinting patterns although some of them carried the same types of SE genes. These data suggest that combinations of esr-mPCR and AP-PCR can provide a powerful approach for epidemiological investigation of toxigenic S. aureus isolates.

Staphylococcus aureus associated with mammary glands of cows: genotyping to distinguish different strains among herds.

The hypothesis that strains of Staphylococcus aureus are more likely to be unique to a herd than common to several herds was tested. Herds (n=28) from nine geographic areas of Korea, with elevated milk somatic cell counts (>500000 cells/ml) were enrolled in this study. Mammary quarter milk samples were aseptically collected from all lactating cows (n=616) with at least three functional quarters. Milk was cultured and S. aureus isolates were typed using pulse field gel electrophoresis of DNA SmaI digests. A total of 181 cows were identified as having S. aureus intramammary infections. A total of 52 different types of S. aureus were identified and 34 (65.4%) were associated with a single herd. A total of 18 types of S. aureus were found in multiple herds; 14 types were found in two herds, and four types were found in three herds. Herds with 1, 2, 3, and more than 3 types, were: four (14.3%); eight (28.6%); nine (32.1%); and seven (25.0%). The data indicate that the majority of strains were found in one herd only, and more than 90% were found in two or less herds, suggesting that strains of S. aureus are more likely to be restricted to a single herd, than found in multiple herds.

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.

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.

Expression of staphylococcal enterotoxin C1 in Escherichia coli.

The structural gene encoding staphylococcal enterotoxin C1 was cloned into Escherichia coli and localized on a 1.5-kilobase HindIII-ClaI DNA fragment by subcloning. The toxin was partially purified from E. coli clones and shown to be immunologically identical to enterotoxin C1 from Staphylococcus aureus. The cloned toxin also had the same molecular weight (26,000) and charge heterogeneity as staphylococcus-derived enterotoxin. Toxins from both sources were equally biologically active.

Conservation of the biologically active portions of staphylococcal enterotoxins C1 and C2.

We determined the primary sequence of staphylococcal enterotoxin (SE) C2 by sequencing its cloned structural gene, entC2. The entC2 structural gene contains an 801-base-pair open reading frame which encodes a 266-amino-acid precursor with a molecular weight of 30,608. Mature SE C2, produced by removal of the signal peptide, contains 239 amino acids with a molecular weight of 27,531. A sequence comparison between SE C2 and SE C1 showed that the 167 carboxyl amino acids in both toxins were 100% conserved. In contrast, the 72 N-terminal residues were 10% divergent. This provides additional evidence that carboxyl regions of staphylococcal and streptococcal pyrogenic toxins determine shared biological activities and cross-reactive epitopes.

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins.

The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27,496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.

Chemical and immunological analysis of the complex structure of Escherichia coli alpha-hemolysin.

Escherichia coli alpha-hemolysin (AH) purified from culture supernatants by gel filtration and ion-exchange chromatography was heterogeneous in charge and size. A 107,000-dalton protein was identified as the product of the hlyA gene by its reactivity with anti-AH monoclonal antibodies. Proteolysis of the product of the hlyA gene occurred but was not required for transport of the protein through the cell wall. Active AH had a larger size and lower pI than analysis of the hlyA gene sequence would predict, thus suggesting that the hlyA protein is complexed with other bacterial products. Lipopolysaccharide was detected in purified hemolysin complex preparations and may be a major component of the complexes. These findings suggest several possible mechanisms for release of AH from the bacterial cell including release by outer membrane fragmentation. The existence of AH complexed with lipopolysaccharide may have important implications in understanding its toxicity.

Composition of affinity-purified alpha-hemolysin of Escherichia coli.

Escherichia coli alpha-hemolysin was purified from culture supernatants by affinity chromatography, using a hemolysis-neutralizing monoclonal antibody ligand. Purified hemolysin contains several proteins and lipopolysaccharides. Thus, alpha-hemolysin exists as a macromolecular complex and may be exported from E. coli cells by outer membrane fragmentation.

Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates.

Staphylococcus aureus may contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates of S. aureus are further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individual S. aureus isolate possesses. We show here, using S. aureus isolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.

Genes encoding staphylococcal enterotoxins G and I are linked and separated by DNA related to other staphylococcal enterotoxins.

Fifteen randomly selected Staphylococcus aureus isolates, known to carry the staphylococcal enterotoxin (SE) G determinant (seg), were shown to carry the SEI determinant (sei). To determine whether these two genes are linked, two S. aureus strains (FRI445 and FRI572), each containing the seg and sei determinants, were further analyzed. In these strains, sei is located 2,002 bp upstream of seg. Within the intergenic nucleotide sequence are three regions of nucleotide sequence with significant identity to the sequences of other SE genes. Characterization of the DNA regions surrounding the seg and sei determinants will allow a better understanding of the association between these two genes and may explain why they are so frequently observed simultaneously in S. aureus isolates.

Cyanobacterial stimulation of growth and oxygen uptake by Legionella pneumophila.

A laboratory-adapted strain of Legionella pneumophila grew in coculture with Fischerella. Insoluble Fischerella slime contained carbohydrate and protein and was isolated from cultures on filters. Slime-free filtrates separated into three 280-nm absorbance peaks on Sephadex G-25. Peaks 1 and 2 contained protein and carbohydrate and stimulated Legionella respiration. Peak 3 and slime had no effect.

Characterization of surfaces involved in adherence of Legionella pneumophila to Fischerella species.

Legionella pneumophila adheres to the slime coat of Fischerella spp. This was shown by microscopic examination and by a decline in L. pneumophila CFU in samples removed from coincubation mixtures of both organisms. Binding of partially purified Fischerella slime by L. pneumophila was most efficient by young, less hydrophobic L. pneumophila cells than by older, more hydrophobic cells. Uptake of crystal violet and partitioning into hexadecane were used to measure hydrophobicity of L. pneumophila. Purified soluble Legionella antigen also bound to Fischerella slime, as shown by indirect immunofluorescence. Adherence was not specific for L. pneumophila, since a variety of gram-negative, gram-positive, and acid-fast bacteria also bound to Fischerella slime.