Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. enzyme-linked immunosorbent assay), MS is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (Protein Standard Absolute Quantification) that uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest. Molecular & Cellular Proteomics 6:2139 -2149, 2007.Mass spectrometry MS has greatly contributed to the maturation of proteomics (1). It is now possible to characterize hundreds of proteins in an hour time frame and compare protein abundances in pairs of samples. The next frontier lies in accurate absolute quantitation. Although label-free spectral counting approaches (2, 3) are attracting considerable interest, robust absolute quantitative methodologies typically rely on the well-established isotope dilution principle (4), in which internal standardization is achieved with isotope-labeled homologs of specific proteolytic peptides from the target protein(s) (5, 6). The Absolute Quantitation (AQUA) 1 peptide strategy uses chemically synthesized isotope-labeled peptides which are spiked into the samples in known quantities before MS analysis (5-8). Recently, the synthesis and metabolic labeling of an artificial concatemer of standard peptides (QconCAT), which can be spiked into the samples before trypsin digestion, was introduced to extend the number of quantified proteins (9 -12). Although AQUA and QconCAT approaches have significantly advanced the quantitative measurement of proteins in biological samples, calibration with AQUA peptides and QconCAT constructs suffer from the following limitations: 1) a failure to take into account the actual efficiency of the proteolysis step required before MS analysis; 2) an incompatibility with sample prefractionation, which is often necessary when dealing with biological samples (13); and 3) a poor protein sequence coverage, limiting the statistical reliability of the quantification. We ...

Methicillin-resistant Staphylococcus aureus is increasingly responsible for staphylococcal infections in the community. A large percentage of the community-acquired methicillin-resistant (CA-MRSA) strains in the USA produce Panton–Valentine leukocidin (PVL), which is associated with severe infections. The virulence of the clinical CA-MRSA strain USA300 was compared to that of its isogenic pvl-deleted mutant, and it was shown that PVL contributes to lung and muscle tissue destruction, respectively, in murine necrotizing pneumonia and skin infection models. Mice infected with the USA300 strain developed a dominant anti-PVL response. The PVL subunits were therefore tested as vaccinogens against this isolate, and their vaccine efficacy correlated with both the route of vaccination and infection. These data suggest that PVL is a virulence factor in murine CA-MRSA infections.

To test the hypothesis that the Staphylococcus aureus enterotoxin gene cluster (egc) can generate new enterotoxin genes by recombination, we analyzed the egc locus in a broad panel of 666 clinical isolates of S. aureus. egc was present in 63% of isolates, confirming its high prevalence. The archetypal organization of the egc locus, consisting of five enterotoxin genes plus two pseudogenes, was found in 409 of 421 egc-positive strains. The egc locus was incomplete in a few strains and occasionally harbored an insertion sequence and transposase genes. These strains may represent evolutionary intermediates of the egc locus. One strain with an atypical egc locus produced two new enterotoxins, designated SElV and SElU2, generated by (i) recombination between selm and sei, producing selv, and (ii) a limited deletion in the ent1-ent2 pseudogenes, producing selu2. Recombinant SElV and SElU2 had superantigen activity, as they specifically activated the T-cell families V␤ 6, V␤ 18, and V␤ 21 (SElV) and V␤ 13.2 and V␤ 14 (SElU2). Immunoscope analysis showed a Gaussian CDR3 size distribution of T-cell receptor V␤ chain junctional transcripts of expanded V␤ subsets in toxin-stimulated cultures, reflecting a high level of polyclonality. These data show that egc is indeed capable of generating new superantigen genes through recombination.Staphylococcus aureus produces a large variety of exotoxins, including staphylococcal enterotoxins A to E (SEA to SEE), SEG to SER, and SEU; staphylococcal enterotoxin-like toxins (SEls); and toxic shock syndrome (TSS) toxin-1 (5, 23). These toxins are responsible for specific acute clinical syndromes such as TSS (due to both TSS toxin-1 SEs and SEls), food poisoning (due to SEs), and staphylococcal scarlet fever (considered a mild form of TSS) (10,26,34).All these toxins share certain structural and biological properties, suggesting that they derive from a common ancestor (16,21). They exhibit superantigen activity, stimulating polyclonal T-cell proliferation through coligation between major histocompatibility complex class II molecules on antigen-presenting cells (APC) and the variable portion of the T-cell antigen receptor ␤ chain or ␣ chain (TCR V␤ and TCR V␣, respectively), with no need for prior APC processing (4,13,21,22,37,39). The pattern of V␤/V␣ activation is specific to each superantigen (4, 12). T-cell/APC activation by these toxins leads to the release of various cytokines/lymphokines and interferon, enhances endotoxic shock, and causes T-and B-cell immunosuppression, all of which may undermine the immune response against bacterial infection (5, 10, 25).All the genes encoding these toxins are harbored by mobile elements, including bacteriophages, pathogenicity islands, genomic islands, and plasmids (10,20,28,36). Only the enterotoxin gene cluster (egc) is organized as an operon, consisting of two enterotoxin genes (seg and sei), three enterotoxinlike genes with proven superantigenic activity but not emetic properties (selo, selm and seln), and two pseudogenes (ent1 and -2). This ...

Enterotoxin A (SEA) might play a key role in sea-positive S. aureus sepsis by triggering over-expression of inflammatory mediators associated with shock. Novel treatments targeting superantigens, especially the sea gene, might be beneficial in the treatment of S. aureus sepsis.

The molecular mechanism of Staphylococcus aureus phathogenicity is complex and involves several toxins, including the famous staphylococcal enterotoxin (SE) and toxic shock syndrome toxin-1 (TSST-1). Although these toxins were discovered in specific clinical contexts of food poisoning and menstrual toxic shock syndrome, they share common biochemical and biological properties. As superantigens they are able to massively activate mononuclear cells and T cells regardless of the antigenic specificity of the T cells. To date, 19 different enterotoxins and related toxins have been described in S. aureus with some differences in structure and biological activity. It has been clearly demonstrated that most human S. aureus isolates harbor at least one gene encoding for these toxins. It is suspected that S. aureus produces SEs and TSST-1 in humans from colonization to infection, whatever the clinical situation. It is proposed that the production of SEs plays a role not only in classical staphylococcal infections but also in noninfectious diseases. This review will focus on recent findings related to staphylococcal superantigens and their impact on human diseases.

PCR was employed to determine the presence of all known superantigen genes (sea, seq, and tst) and of the exotoxin-like gene cluster (set) in 40 Staphylococcus aureus isolates from blood cultures and throat swabs; 28 isolates harbored superantigen genes, five on average, and this strictly correlated with their ability to stimulate T-cell proliferation. In contrast, the set gene cluster was detected in every S. aureus strain, suggesting a nonredundant function for these genes which is different from T-cell activation. No more than 10% of normal human serum samples inhibited the T-cell stimulation elicited by egc-encoded enterotoxins (staphylococcal enterotoxins G, I, M, N, and O), whereas between 32 and 86% neutralized the classical superantigens. Similarly, intravenous human immunoglobulin G preparations inhibited egc-encoded superantigens with 10-to 100-fold-reduced potency compared with the classical enterotoxins. Thus, there are surprisingly large gaps in the capacity of human serum samples to neutralize S. aureus superantigens.Staphylococcus aureus persists as a commensal microorganism in 10 to 30% of the population, but the organism is also a common cause of food poisoning and infections of different severity such as skin abscesses and wound infections, osteomyelitis, endocarditis, pneumonia, toxic shock syndrome, and staphylococcal scarlet fever (21). S. aureus is one of the most frequent causes of hospital-acquired infections, and the emergence and spread of multiresistant strains give rise to concern. The pathogenicity of S. aureus is multifactorial, and the versatility of this organism is underscored by recent clinical studies (4,14,16,31,37,38).Superantigens activate large subpopulations of T lymphocytes by directly cross-linking certain T-cell receptor V␤ domains with conserved structures on major histocompatibility complex class II molecules (32). They belong to the most potent T-cell mitogens known and can induce massive systemic cytokine release, leading to the symptoms of toxic shock syndrome (22). Among the virulence factors of S. aureus are the staphylococcal enterotoxins, the causative agents of food poisoning. They also act as superantigens. Whole-genome sequencing of several S. aureus clinical isolates has revealed that all 17 known staphylococcal enterotoxins (staphylococcal enterotoxins A to E and G to Q and toxic shock syndrome toxin 1) are encoded on mobile genetic elements together with other virulence factors (3,18,40). For example, the recently described enterotoxin gene cluster egc, which contains the five superantigen genes seg, sei, sem, sen, and seo, as well as two pseudogenes is located on the genomic island SaPI3 (18).egc is special in that it functions as an operon and its genes are transcribed into a single polycistronic mRNA (13). In addition, a large cluster of up to 11 genes with sequence homology to superantigens has been discovered on the genomic island SaPI2; they have been termed staphylococcal exotoxinlike genes, or set (3,10,18,39). For an overview of the organization ...

Staphylococcus aureus can produce a wide variety of exotoxins, including toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxins, and staphylococcal enterotoxin-like toxins. These toxins share superantigenic activity. To investigate the ␤ chain (V␤) specificities of each of these toxins, TSST-1 and all known S. aureus enterotoxins and enterotoxin-like toxins were produced as recombinant proteins and tested for their ability to induce the selective in vitro expansion of human T cells bearing particular V␤ T-cell receptors (TCR). Although redundancies were observed between the toxins and the V␤ populations, each toxin induced the expansion of distinct V␤ subsets, including enterotoxin H and enterotoxin-like toxin J. Surprisingly, the V␤ signatures were not associated with a specific phylogenic group of toxins. Interestingly, each human V␤ analyzed in this study was stimulated by at least one staphylococcal superantigen, suggesting that the bacterium derives a selective advantage from targeting the entire human TCR V␤ panel.Staphylococcus aureus produces a broad range of exoproteins, including staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1) (9). These toxins were initially implicated in staphylococcal food poisoning (SEs) and TSS (TSST-1) (39). Since the first characterization of SEA and SEB in 1959 to 1960 by Casman and Bergdoll, 18 different SEs have been described; they are designated SEA to SEV, in the chronological order of their discovery (2,5,41). Some were renamed SE-like toxins (SEl), because either no emetic properties were detected or because they were not tested in primate models (21, 41).SEs, SEls, and TSST-1 share certain structural and biological properties. They have similar sizes (23 to 29 kDa), and their crystal structures, established for SEA, SEB, SEC, SED, SEH, SElI, SElK, and TSST-1, reveal significant homology in their secondary and tertiary conformations (26).

The severity of Staphylococcus aureus sepsis is positively associated with staphylococcal enterotoxin A (SEA) and negatively associated with the enterotoxin gene cluster (egc), which encodes five staphylococcal enterotoxins. We postulated that the variable, clinical severity of S. aureus sepsis might be a result of differences in the inflammatory properties of staphylococcal superantigens. We therefore compared the inflammatory properties of SEA with those of staphylococcal entérotoxin G (SEG), a member of the five egc superantigens. We found that SEA and SEG had similar superantigenic properties, as they induced CD69 expression on T lymphocytes and selective expansion of Vbeta subpopulations. Contrary to SEG, however, SEA induced a strong proinflammatory/Th1 response, including TNF-alpha and MIP-1alpha production. These results suggest that the association of SEA with the severity of S. aureus septic shock, characterized by a deleterious, inflammatory cascade, may be explained partly by the specific proinflammatory properties of this superantigen.