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Background: Staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic shock syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus, belong to the subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T cells bearing specific variable regions of the T cell receptor beta chain (Vbeta). Quantitative real time PCR (qRT-PCR) has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vbeta subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing to selected Vbeta nucleotide sequences and SYBR Green I reporter has not been applied to assess Vbeta-dependent expansion of T cells by SAgs.Methods: Human peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vbeta subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vbeta nucleotide sequences.Results: qRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies, standard curves were generated for each primer set. The average slope and R2 of standard curves were -3.3764 +/- 0.0245 and 0.99856 +/- 0.000478, respectively, demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions, SAg Vbeta specificities observed in this study were similar to those reported in previous studies.Conclusions: The qRT-PCR method established in this study produced an accurate and reproducible assessment of Vbeta-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool in the characterization T cell proliferation by newly discovered SAg and in the investigation of biological effects of SAgs linked to pathogenesis. [ABSTRACT FROM AUTHOR]

Maintenance of phospholipid (PL) and lipopoly- or lipooligosaccharide (LPS or LOS) asymmetry in the outer membrane (OM) of Gram-negative bacteria is essential but poorly understood. The Yersinia pestis OM Ail protein was required to maintain lipid homeostasis and cell integrity at elevated temperature (37°C). Loss of this protein had pleiotropic effects. A Y. pestis Δail mutant and KIM6 + wild type were systematically compared for (i) growth requirements at 37°C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernatants, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild-type phenotype. The Δail mutant grew normally at 28°C but lysed at 37°C when it entered stationary phase, as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immunofluorescence microscopy showed that the Δail mutant did not assemble Caf1 capsule. Expression of heat shock promoter rpoE or rpoH fused to a lux operon reporter were not induced when the Δail mutant was shifted from 28°C to 37°C ( P  < 0.001 and P  < 0.01, respectively). Mutant lysis was suppressed by addition of 11 mM glucose, 22 or 44 mM glycerol, 2.5 mM Ca 2+ , or 2.5 mM Mg 2+ to the growth medium or by a mutation in the phospholipase A gene ( pldA ::miniTn 5 , ΔpldA , or PldA S164A ). A model accounting for the temperature-sensitive lysis of the Δail mutant and the Ail-dependent stabilization of the OM tetraacylated LOS at 37°C is presented. IMPORTANCE The Gram-negative pathogen Yersinia pestis transitions between a flea vector (ambient temperature) and a mammalian host (37°C). In response to 37°C, Y. pestis modifies its outer membrane (OM) by reducing the fatty acid content in lipid A, changing the outer leaflet from being predominantly hexaacylated to being predominantly tetraacylated. It also increases the Ail concentration, so it becomes the most prominent OM protein. Both measures are needed for Y. pestis to evade the host innate immune response. Deletion of ail destabilizes the OM at 37°C, causing the cells to lyse. These results show that a protein is essential for maintaining lipid asymmetry and lipid homeostasis in the bacterial OM.

Yersinia pestis grown with physiologic glucose increased cell autoaggregation and deposition of extracellular material, including membrane vesicles. Membranes were characterized, and glucose had significant effects on protein, lipid, and carbohydrate profiles. These effects were independent of temperature and the biofilm-related locus pgm and were not observed in Yersinia pseudotuberculosis.

A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1(+)) Pgm(-) (pigmentation negative)/, KIM6(pCD1(-)) Pgm(+), and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 Δilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). Following intravenous challenge with Y. pestis KIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system (in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 Δilp had a 55-fold increase in the 50% lethal dose ([LD(50)] 1.64 × 10(4) CFU) compared to the parental wild-type strain LD(50) (2.98 × 10(2) CFU). These findings identified Ilp as a novel virulence factor of Y. pestis.

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone.

A previously beach-stranded, juvenile, male, bottlenose dolphin (Tursiops truncatus) was diagnosed with vertebral osteomyelitis of unknown etiology. Antemortem serological testing suggested past or current Brucella sp. infection; however, this could not be confirmed prior to death despite multiple isolation attempts from aspirates, blood, and biopsies. Systemic antibiotics were administered for over a year to control the suspected infection; however, the animal succumbed peracutely to infection by a highly pathogenic, enterotoxin-secreting Staphylococcus sp. Gross necropsy findings included a fistulous tract leading to locally extensive osteomyelitis of a coccygeal vertebra with sequestra and osteophytes from which a Brucella species was isolated. Histopathological examination of intestine revealed pseudomembranous enteritis with a uniform population of intraluminal Gram-positive cocci. Staphylococcus aureus was isolated in pure culture from the intestine and tested positive for the staphylococcal enterotoxin A gene by polymerase chain reaction analysis. Serum taken shortly before death had endotoxin and elevated antibody titers to staphylococcal enterotoxin A when compared to samples collected during a period of apparent good health 18 months earlier. The isolation of a pyrogenic toxin superantigen-producing staphylococcal isolate, clinical signs, and diagnostic findings in this animal resembled some of those noted in human toxic shock syndrome. The present case highlights the clinical challenges of treating chronic illnesses, complications of long-term antibiotic use, and promotion of pathogenic strains in cases of prolonged rehabilitation of marine mammals.

Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage.

The coagulase-negative staphylococci (CNS) are the most prevalent mastitis pathogen group yet their virulence characteristics have not been well described. We investigated the presence of 19 classical and newly described staphylococcal superantigen (SAg) genes in CNS isolates from bovine intramammary infections (IMI). A total of 263 CNS representing 11 different Staphylococcus spp. were examined, and 31.2% (n=82) of CNS isolates had one or more SAg genes; there were 21 different SAg gene combinations. The most prevalent combination of SAg genes (seb, seln and selq; n=45) was found in S. chromogenes, S. xylosus, S. haemolyticus, S. sciuri subsp. carnaticus, S. simulans and S. succinus. The genes for SAgs appear to be widely distributed amongst CNS isolated from bovine IMI., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0% (200/263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0% (255/263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a prominent human pathogen and a leading cause of community- and hospital-acquired bacterial infections worldwide. Herein, we describe the identification and characterization of the S. aureus 67.6-kDa hypothetical protein, named for the surface factor promoting resistance to oxidative killing (SOK) in this study. Sequence analysis showed that the SOK gene is conserved in all sequenced S. aureus strains and homologous to the myosin cross-reactive antigen of Streptococcus pyogenes. Immunoblotting and immunofluorescence analysis showed that SOK was copurified with membrane fractions and was exposed on the surface of S. aureus Newman and RN4220. Comparative analysis of wild-type S. aureus and an isogenic deletion strain indicated that SOK contributes to both resistance to killing by human neutrophils and to oxidative stress. In addition, the S. aureus sok deletion strain showed dramatically reduced aortic valve vegetation and bacterial cell number in a rabbit endocarditis model. These results, plus the suspected role of the streptococcal homologue in certain diseases such as acute rheumatic fever, suggest that SOK plays an important role in cardiovascular and other staphylococcal infections.

Yersinia pestis, the causative agent of plague, is one of the most virulent microorganisms known. The outer membrane protein X (OmpX) in Y. pestis KIM is required for efficient bacterial adherence to and internalization by cultured HEp-2 cells and confers resistance to human serum. Here, we tested the contribution of OmpX to disease progression in the fully virulent Y. pestis CO92 strain by engineering a deletion mutant and comparing its ability in mediating pneumonic plague to that of the wild type in two animal models. The deletion of OmpX delayed the time to death up to 48 h in a mouse model and completely attenuated virulence in a rat model of disease. All rats challenged with 1 × 10(8) CFU of the ompX mutant survived, compared to the 50% lethal dose (LD50) of 1.2 × 10(3) CFU for the wild-type strain. Because murine serum is not bactericidal for the ompX mutant, the mechanism underlying the delay in time to death in mice was attributed to loss of adhesion/internalization properties but not serum resistance. The rat model, which is most similar to humans, highlighted the critical role of serum resistance in disease. To resolve conflicting evidence for the role of Y. pestis lipopolysaccharide (LPS) and OmpX in serum resistance, ompX was cloned into Escherichia coli D21 and three isogenic derivatives engineered to have progressively truncated LPS core saccharides. OmpX-mediated serum resistance, adhesiveness, and invasiveness, although dependent on LPS core length, displayed these functions in E. coli, independently of other Yersinia proteins and/or LPS. Also, autoaggregation was required for efficient OmpX-mediated adhesiveness and internalization but not serum resistance.

Biofilms are surface-associated communities of microbes encompassed by an extracellular matrix. It is estimated that 80% of all bacterial infections involve biofilm formation, but the structure and regulation of biofilms are incompletely understood. Extracellular DNA (eDNA) is a major structural component in many biofilms of the pathogenic bacterium Staphylococcus aureus, but its role is enigmatic. Here, we demonstrate that beta toxin, a neutral sphingomyelinase and a virulence factor of S. aureus, forms covalent cross-links to itself in the presence of DNA (we refer to this as biofilm ligase activity, independent of sphingomyelinase activity) producing an insoluble nucleoprotein matrix in vitro. Furthermore, we show that beta toxin strongly stimulates biofilm formation in vivo as demonstrated by a role in causation of infectious endocarditis in a rabbit model. Together, these results suggest that beta toxin cross-linking in the presence of eDNA assists in forming the skeletal framework upon which staphylococcal biofilms are established.

Background: The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined., Methodology/principal Findings: In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5., Conclusions: Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.

Although many effects of staphylococcal superantigens (SAg) on T cells are well established, less is known about their effects on APC. In this study, bovine PBMC were stimulated with a low dose of staphylococcal enterotoxin C1 (SEC1). The phenotype of adherent cells (Ac) derived from bovine PBMC cultured with SEC1 [SEC1-stimulated Ac (sAc)] for 192 h was CD14(-), CD68(-), CD163(-), dendritic cell (DC)-specific ICAM-3-grabbing nonintegrin(+), MHC class II (MHC II)(high), CD11a(low), CD11b(high), CD11c(high), and CD1b(high), suggesting these cells were dendritic cells (DC). SEC1 also induced transcription of the CXCL1, -2, and -3 family, CXCL6, CCL2, and CCL5 genes in sAc, which increased rapidly but returned to basal levels by 48 h. In contrast, increased transcription of CCL3, CCL8, and CXCL12, responsible for mononuclear cell migration and chronic inflammation, was sustained. In vitro cell migration assays showed vigorous migration of granulocytes, followed by migration of mononuclear cells. The autologous MLR showed that sAc induced a dose-dependent proliferation of CD4(+) T cells and an even stronger proliferation of CD8(+) T cells. This effect was inhibited or reduced by pretreatment with mAb to CD11b, MHC II, or MHC II plus CD18. These results indicate that stimulation of bovine PBMC by SAg induces differentiation of monocytes into DC.

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.

HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

An effective intranasal (i.n.) vaccine against pneumonic plague was developed. The formulation employed two synthetic lipid A mimetics as adjuvant combined with Yersinia pestis-derived V- and F1-protective antigens. The two nontoxic lipid A mimetics, classed as amino-alkyl glucosaminide 4-phosphates (AGPs) are potent ligands for the Toll-like receptor (TLR) 4. Using a murine (BALB/c) pneumonic plague model, we showed a single i.n. application of the vaccine provided 63% protection within 21 days against a Y. pestis CO92 100 LD50 challenge. Protection reached 100% by 150 days. Using a homologous i.n. 1 degrees /2 degrees dose regimen, with the boost administered at varying times, 63% protection was achieved within 7 days and 100% protection was achieved by 21 days after the first immunization. Little or no protection was observed in animals that received antigens alone, and no protection was observed when the vaccine was administered to BALB/c TLR4 mutant mice. Vaccine-induced serum IgG titers to F1 and V-antigen were reflected in high titers for IgG1 and IgG2a, the latter reflecting a bias for a cell-mediated (TH1) immune response. This intranasal vaccine showed 90% protection in Sprague-Dawley rats challenged with 1000 LD50. We conclude that lipid A mimetics are highly effective adjuvants for an i.n. plague vaccine.

This study analysed the effect of priming the innate immune system using synthetic lipid A mimetics in a Yersinia pestis murine pulmonary infection model. Two aminoalkyl glucosaminide 4-phosphate (AGP) Toll-like receptor 4 (TLR4) ligands, delivered intranasally, extended time to death or protected against a lethal Y. pestis CO92 challenge. The level of protection was dependent upon the challenge dose of Y. pestis and the timing of AGP therapy. Protection correlated with cytokine induction and a decreased bacterial burden in lung tissue. AGP protection was TLR4-dependent and was not evidenced in transgenic TLR4-deficient mice. AGP therapy augmented with subtherapeutic doses of gentamicin produced dramatically enhanced survival. Combined, these results indicated that AGPs may be useful in protection of immunologically naive individuals against plague and potentially other infectious agents, and that AGP therapy may be used synergistically with other therapies.

Beta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-A resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.

The goal of this study was to characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis, Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6(+) parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 degrees C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro. These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.

Silica nanowires (NWs) were used to introduce the Shiga toxin type 1 A subunit (StxA1) into cultured bovine and human epithelial cells. We extended technology developed in our laboratories that employs fibronectin (Fn) to induce integrin-mediated uptake of NWs by coating NWs with StxA1 and Fn. The bonding strengths of Fn and StxA1 to the surface of NWs were measured by X-ray photoelectron spectroscopy. This technique demonstrated complex interactions between Fn, StxA1, and the NWs. Neutral red cytotoxicity assays and field emission scanning electron microscopy confirmed that the NW-StxA1-Fn complexes were effectively internalized and caused cell death. This indicates that NWs can carry StxA1 and potentially other toxic or therapeutic agents into eukaryotic cells. Ongoing studies include improved functionalizing of NWs aimed at increasing internalization efficiency and substituting ligands for specific cell targeting.

Regulatory T cells (T(regs)) help control the development and maintenance of protective immunity and can lead to aberrant immune responses to some pathogens. Several lines of evidence suggest that T(regs) are induced by exposure to superantigens (SAgs) in vitro or in vivo. In this study, bovine peripheral blood mononuclear cells (PBMC) were exposed in vitro to a relatively low dose (5 ng/ml) of staphylococcal enterotoxin C1 (SEC1) for up to 10 days. Upon stimulation, CD4+ and CD8+ T cells initially proliferated at similar rates. Subsequently, from days 6 through 10, most CD4+ and CD8+ T cells proliferated regardless of Vbeta specificity, but the proliferation of CD8+ T cells occurred more vigorously. The transcription of CD25 and CD152 genes increased, whereas that of interleukin-2 (IL-2) decreased. gammadelta T cells appeared to be unresponsive. An increase in the transcription of IL-10 and transforming growth factor beta (TGF-beta) genes in SEC1-stimulated cultures was attributed to the CD4+ CD25+ T-cell subpopulation. The expression of Foxp3 mRNA also increased and was accompanied by the upregulation of CD152 and the downregulation of IL-2 transcription, suggesting that cells in this subpopulation are T(regs). Functionally, SEC1-stimulated CD4+ T cells suppressed the proliferation of naive PBMC in response to heat-killed-fixed Staphylococcus aureus. The suppression was partially mediated by IL-10 and TGF-beta, another characteristic of certain types of T(regs.) The CD8+ T-cell population also suppressed naive PBMC through another mechanism not mediated by IL-10 or TGF-beta. These results provide further insight into the potential mechanisms by which SAgs could contribute to evasion of the immune response, affecting the outcome of infection or colonization.

We previously demonstrated that stimulation of bovine peripheral blood mononuclear cells (PBMCs) with staphylococcal enterotoxin C (SEC), led to an inversion of the CD4(+):CD8(+)T cell ratio and generation of an atypical CD8(+)T cell subpopulation expressing CD26. In the present study, we examined T cell apoptosis and proliferation profiles of PBMC subpopulations in cultures stimulated with SEC. Unlike when stimulated with concanavalin A, nucleic acid synthesis in bovine PBMC cultures stimulated with SEC was low during the first four days but increased greatly on day 5. In contrast, nucleic acid synthesis in human PBMC cultures stimulated with SEC increased continuously. To investigate the mechanism of delayed bovine T cell proliferation, various cell phenotypes were monitored. The inversion of the bovine CD4(+):CD8(+)T cell ratio in PBMC cultures stimulated by SEC was associated with higher proliferation and lower apoptosis of CD8(+)T cells compared to CD4(+)T cells. The mRNA levels for interleukin (IL)-4 and IL-13 were sustained over 4 days but IL-12 mRNA levels dropped to background on day 2. These data suggest that SEC induces a prolonged Th-2- biased microenvironment, and together with the inversion of the bovine CD4(+):CD8(+)T cell ratios in bovine PBMC cultures with SEC, may in part explain the inability of the mammary immune system to establish an effective response to Staphylococcus aureus infections.

Neisseria meningitidis groups A (GAM) and W135 capsular polysaccharides (CPs) were bound to recombinant Staphylococcus aureus enterotoxin C1 (rSEC). The CPs were activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate and then bound to adipic acid dihydrazide derivatives of rSEC. Syntheses were conducted with native GAM CP (GAMP), W135 CP (W135P), and ultrasonicated or hydrazine-treated W135P at various concentrations of reactants, pHs, and ionic strengths. The conjugates were characterized by compositional and serologic analyses, high-performance size-exclusion chromatography with multi-angle laser light scattering detection, and immunogenicity in 5- to 6-week-old mice. Conjugates injected subcutaneously in phosphate-buffered saline elicited immunoglobulin G (IgG) responses against their respective CPs and rSEC, whereas GAMP and W135P alone did not induce detectable CP antibodies. The O-acetyl content of W135P was low, and its removal had no adverse effect upon the conjugate's immunogenicity. Reduction of the molecular size of W135P by treatment with hydrazine improved the immunogenicity of W135P-rSEC. IgG anti-CP elicited by the conjugates showed complement-dependent bactericidal activity against their respective organisms, and IgG anti-rSEC neutralized the T-cell proliferative activity of native SEC. A bivalent formulation of GAMP-rSEC and W135P-rSEC elicited IgG anti-CP at comparable levels to those induced by the conjugates administered separately.

Our recent study has provided that the in vitro SEC-induced proliferation of bovine T cells is preceded by a period of a non-proliferative immunoregulation of T cells that may be associated with cytokine production regulated by type 1 or type 2 T cells. Inversion of CD4(+):CD8(+) T cell ratio and induction of CD8(+) T cells with immunoregulatory activity could increase the probability of intracellular survival of Staphylococcus aureus (S. aureus). The increase of activated CD8(+)(ACT2(+) BoCD8(+)) T cells in cows with mastitis caused by S. aureus may be associated with immune-regulatory function in the bovine mammary gland. The difference and similarity between bovine activated CD8(+) T cells (CD8(+)CD26(+)) and well-established human CD4(+)CD25(+) T regulatory (Tr) cells may help to reveal their unique immune regulatory system in the host infected with S. aureus.

In recent years several new staphylococcal enterotoxins (SEs) have been described, which currently have largely unknown frequencies of occurrence and roles in human or animal disease. One hundred and ninety-one Staphylococcus aureus isolates from cows (99), goats (39), sheep (23), rabbits (15), chickens (15) and a cat (1) were screened for SE genes sea-see, seg-seo and seq and for the tst gene encoding staphylococcal toxic shock syndrome toxin-1 using multiplex PCRs and individual PCRs for the seb and sek genes. One hundred and ten isolates tested positive for at least one of these 16 superantigen (SAg)-encoding genes. There were statistically significant differences in the frequencies of some of these SAg genes between isolates from different animals. No strain possessed either the sea or see gene. The sec gene was present in 51 isolates, the sed gene in eight and the seb gene in one. The seh gene was found in four strains and the sek and seq genes together in one isolate. The most common combinations of genes were the egc cluster, bearing the seg, sei, sem, sen and seo genes, in 47 isolates, the sec, sel and tst gene combination typical of the SaPIbov pathogenicity island in 44 isolates, the egc cluster lacking the seg gene in 11 isolates, the sed and sej genes in nine isolates, and the sec and tst genes without the sel gene in seven isolates. The higher frequencies of the sec and tst genes together and the lower frequencies of the egc gene cluster among the SAg gene-positive sheep or goat isolates compared to bovine isolates were statistically significant. Of 36 bovine isolates that were mitogenic for human T lymphocytes, four were negative for the 16 SAg genes tested for, while a further 14 gave borderline results in the mitogenicity assay, 12 of which were SAg gene-negative. Twenty-nine strains lacking all the SAg genes did not induce T-cell proliferation. This survey indicates that novel SE genes seg, sei, sel, sem, sen and seo along with the sec and tst genes predominate in S. aureus from animal hosts. The mitogenicity assays indicate that further uncharacterized SAgs may be present in bovine isolates.

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.

The International Nomenclature Committee for Staphylococcal Superantigens proposes an international procedure for the designation of newly described superantigens and putative superantigens, a procedure that will be compatible with the new age of genomics.

Staphylococcus aureus invades a variety of mammalian cells and escapes from the endosome to multiply in the cytoplasm. We had previously hypothesized that the molecular events leading to escape of S. aureus from the endosome involved the Agr virulence factor regulatory system. In this report we demonstrate that temporal changes in intracellular activation of the Agr regulon correlates with expression of membrane active toxins. Also, the initial expression of Agr by even small numbers of staphylococci resulted in the permeabilization of the endosomal membrane and the eventual escape of bacteria into the cytoplasm by 3 h post invasion. After Agr downregulation, a second peak of expression coincided with increased permeability of the host cell membrane. In contrast to the parental strain, an Agr-mutant was unable to escape into the cytoplasm and was observed in intact endosomes as late as 5 h post invasion. These data provide evidence that staphylococcal virulence factor production during invasion of host cells is mediated by an Agr-dependent process that is most accurately described in the context of diffusion sensing.

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.

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.

Staphylococcal enterotoxins are superantigen exotoxins that mediate food poisoning and toxic shock syndrome in humans. Despite their structural and functional similarities, superantigens display subtle differences in biological properties and modes of receptor binding as a result of zinc atoms bound differently in their crystal structures. For example, the crystal structures of the staphylococcal enterotoxins in the type C serogroup (SECs) contain a zinc atom coordinated by one aspartate and two histidine residues from one molecule and another aspartate residue from the next molecule, thus forming a dimer. This type of zinc ligation and zinc-mediated dimerization occurs in several SECs, but not in most other staphylococcal enterotoxin serogroups. This prompted us to investigate the potential importance of zinc in SEC-mediated pathogenesis. Site-directed mutagenesis was used to replace SEC zinc binding ligands with alanine. SEC mutants unable to bind zinc did not have major conformational alterations although they failed to form dimers. Zinc binding was not essential for T cell stimulation, emesis, or lethality although in general the mutants were less pyrogenic. Thus the zinc atom in SECs might represent a non-functional heavy atom in an exotoxin group that has diverged from related bacterial toxins containing crucial zinc atoms.

In this report, we describe the cloning, sequencing, and expression of the bovine orthologue of CD26 (BoCD26). Several monoclonal antibodies specific for a molecule, activation molecule 3 (ACT3), aberrantly expressed on superantigen-stimulated bovine CD8(+) lymphocytes, reacted with recombinant BoCD26 expressed in COS-7 and CHO cells. We also showed that human CD8(+) T cells stimulated by a superantigen expressed CD26 at high levels. These results demonstrate that ACT3 is identical to BoCD26 and suggest that CD26 upregulation on CD8(+) T cells is a general phenomenon of superantigens and not limited to their effects on bovine cells.

Most CD8(+) T cells in cultures of bovine mononuclear cells stimulated with staphylococcal enterotoxin C1 develop an unusual phenotype characterized by expression of activation molecule 3 (ACT3). This superantigen-dependent phenotype may be relevant to immunopathogenesis mediated by certain microbial toxins. The size and N-terminal sequence of immunoprecipitated ACT3 indicate that ACT3 is the bovine orthologue of CD26.

Staphylococcal enterotoxin C (SEC), a superantigen, is the most frequently expressed enterotoxin by bovine strains of Staphylococcus aureus causing mastitis. To examine the possible impact of SEC on the immune response of the bovine mammary gland, we monitored changes in lymphocyte subpopulations in mammary glands of four lactating cows after intramammary instillation of S. aureus strain Rn4220 transformed with a plasmid containing a gene coding for SEC1. Four other lactating cows received the same strain transformed with the plasmid without the SEC1 gene (positive control), and four cows were untreated (negative control). Mammary quarter milk samples for somatic cell count (SCC) analysis and determination of N-acetyl-beta-D-glucosimindase (NAGase) activity levels were collected daily for 21 d postinstillation. Flow cytometry utilizing three-color analysis was used to phenotype lymphocyte subpopulations isolated from milk samples collected on d 0, 4, 7, 11, 14, 18, and 21 postinstillation from all the cows. Milk from mammary gland halves (positive control and experimental) or all mammary quarters (negative control) was collected for flow cytometric analysis. Increased NAGase activity, SCC, and isolated S. aureus demonstrated that infection was established in mammary quarters intrammarily instilled with bacteria. There were no significant differences (P > 0.05) in the proportions of BoCD4 helper T lymphocytes or BoCD8 cytotoxic T lymphocytes between the two infected treatment groups. There was a significant day x treatment difference of the proportion of a gammadelta T cell subpopulation that did not express BoCD2, but did express the ACT2 activation molecule and a significant treatment difference of a gammadelta T cell subpopulation that expressed BoCD2, but not the ACT2 activation molecule (P < 0.05). Results do not support the hypothesis that the presence of the gene for SEC1 alters the mammary BoCD4 or BoCD8 T lymphocyte response to infection.

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.

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.

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.

Previous studies have demonstrated that a proportion of Staphylococcus aureus isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (tst), the SEC bovine variant (sec-bovine), and a gene (sel) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3' end of the GMP synthase gene (gmps) in the S. aureus chromosome. SaPIbov contains a central region of sequence identity with the previously characterized tst pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527-543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of S. aureus strain RF122 were constructed by allele replacement. In vitro bovine Vbeta-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of tst and sec-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.

We reported previously that internalization of Staphylococcus aureus by nonprofessional phagocytes involves an interaction between fibronectin (Fn) binding protein (FnBP) and the host cell, resulting in signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach, Infect. Immun. 67:4673-4678, 1999). The goal of the present study was to identify the host molecules responsible for uptake of the organism through an interaction with FnBP. First, Fn was required for internalization. Addition of small amounts of exogenous Fn stimulated the uptake of S. aureus by HEp-2 cells, which are deficient in Fn synthesis. Fn antibodies blocked internalization of the organism by MAC-T cell monolayers, a bovine epithelial cell line which expresses Fn. Second, a monoclonal antibody (MAb) specific for beta(1) integrins dramatically reduced S. aureus invasion, suggesting that the formation of a Fn bridge linking the host cell beta(1) integrin and FnBP precedes internalization. However, ligand blotting of cell membrane proteins with a functional fragment of FnBP consistently identified an additional approximately 55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as heat shock protein 60 (Hsp60). The interaction between FnBP and Hsp60 also occurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell membrane. Pretreatment of epithelial cells with a MAb specific for eukaryotic Hsp60 significantly reduced internalization of S. aureus. Combined, these results suggest that the FnBP binds directly to both Hsp60 and Fn and is linked to beta(1) integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, beta(1) integrins and Hsp60, suggests that FnBP is a multifunctional adhesin that mediates internalization in a manner similar to that proposed for OpaA, the Neisseria gonorrhoeae FnBP homolog (J. P. M. van Putten, T. D. Duensing, and R. L. Cole, Mol. Microbiol. 29:369-379, 1998).

Staphylococcus aureus and Streptococcus pyogenes express pyrogenic toxin superantigens (PTSAgs) that are associated with toxic shock syndrome (TSS) and staphylococcal food poisoning (SFP). Most PTSAgs cause TSS in deep-tissue infections, whereas only TSS toxin 1 (TSST-1) is associated with menstrual, vaginal TSS. In contrast, SFP has been linked only with staphylococcal enterotoxins (SEs). Because of the differential abilities of PTSAgs to cause systemic or localized symptoms in a site-dependent manner, the present study was undertaken to assess the toxins' abilities to cross mucosal barriers. The activity of three PTSAgs when delivered orally, vaginally, or intravenously to rabbits and orally to monkeys was investigated. TSST-1 induced shock via all three routes in rabbits. Although active when administered intravenously, SEC1 and streptococcal pyrogenic exotoxin A (SPEA) did not cause symptoms when administered orally or vaginally. Only SEC1 induced emesis in the monkey feeding assay. TSST-1, albeit less stable than SEC1 and SPEA to pepsin, induced diarrhea in monkeys. Our results may explain the unique association of TSST-1 with menstrual TSS and why SPEA is only rarely associated with TSS after pharyngitis, despite being highly associated with TSS after subcutaneous infections. Finally, our studies indicate that enterotoxicity in SFP is not the result of superantigenicity.

In this study, we demonstrate that the mechanism by which Staphylococcus aureus induces apoptosis in bovine mammary epithelial (MAC-T) cells involves caspases 8 and 3, two key components of a proteolytic cascade leading to apoptosis. In addition, internalized S. aureus induces expression of the inflammatory cytokines tumor necrosis factor alpha and interleukin-1beta by MAC-T cells. These data suggest that the internalization of S. aureus could induce specific cellular responses in vivo that may ultimately impact the course of infection.

One virulence determinant of Staphylococcus aureus is Beta-toxin, a 37 Kd magnesium-dependent sphingomyelinase C. This toxin lyses erythrocytes (RBCs) containing sphingomyelin in the outer lipid layer of their plasma membrane. Although membranes of both human polymorphonuclear leukocytes (PMNs) and lymphocytes (MNLs) contain small amounts of sphingomyelin, the effect of Beta-toxin on these cells remains controversial. The purpose of this study was to investigate the hemolytic activity of this toxin on RBCs of various species and determine the leukotoxic nature on several types of human leukocytes. One nanogram of Beta-toxin lysed 115,000 sheep erythrocytes (sRBCs) and 82,000 human erythrocytes (hRBCs) in a 'hot-cold' assay and caused cytotoxicity to 325 PMNs and MNLs. Both hemolytic and leukotoxic activity were found to be magnesium-dependent. RBC susceptibility to Beta-toxin correlated with the reported sphingomyelin content of each species. Scanning electron microscopy (SEM) demonstrated that 'hot-cold' incubation with Beta-toxin in the presence of magnesium caused significant morphological changes in the surface structure of both RBCs and PMNs. The changes included the formation of pits and membrane invaginations in the RBCs. The PMNs lost their ruffled membrane appearance and showed overall membrane disintegration. This study demonstrated that the viability of sphingomyelin-containing PMNs and MNLs was significantly decreased by the addition of Beta-toxin, indicating that this toxin does, in fact, have a leukotoxic nature. Leukocytes did not have significant membrane invaginations unlike toxin-treated RBCs; therefore, it is possible that leukotoxicity does not result from membrane lysis.

The staphylococcal exfoliative toxins (ETs) A and B (ETA and ETB) are 27-kDa exotoxins produced by certain strains of Staphylococcus aureus and are the causative agents of staphylococcal scalded-skin syndrome. The crystal structures of the ETs strongly indicate that the proteins are members of the serine protease family of enzymes, although protease activity until now has not yet been conclusively demonstrated. Here, we show that the peptide beta-melanocyte-stimulating hormone (beta-MSH) is cleaved by ETA and that both ETA and ETB are capable of cleaving alpha-MSH. Both toxins exhibit cleavage at specific glutamic acid residues in MSH peptides. Moreover, biologically inactive mutants of ETA were incapable of cleaving beta-MSH.

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.

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.