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Menstrual toxic shock syndrome (mTSS) is a rare life-threatening febrile illness that occurs in women using intravaginal menstrual protection. It is caused by toxic shock syndrome toxin 1 (TSST-1) produced by Staphylococcus aureus , triggering a sudden onset of rash and hypotension, subsequently leading to multiple organ failure. Detecting TSST-1 and S. aureus virulence factors in menstrual fluid could accelerate the diagnosis and improve therapeutic management of mTSS. However, menstrual fluid is a highly complex matrix, making detection of bacterial toxins challenging. Here, we present a mass-spectrometry-based proteomics workflow for the targeted, quantitative analysis of four S. aureus superantigenic toxins in menstrual fluids (TSST-1, SEA, SEC, and SED). This method was applied to characterize toxin levels in menstrual fluids collected from patients with mTSS and healthy women. Toxins were detectable in samples from patients with mTSS and one healthy donor at concentrations ranging from 0 to 0.46 µg/mL for TSST-1, and 0 to 1.07 µg/mL for SEC. SEA and SED were never detected in clinical specimens, even though many S. aureus strains were positive for the corresponding genes. The method presented here could be used to explore toxin production in vivo in users of intravaginal devices to improve the diagnosis, understanding, and prevention of mTSS.

Staphylococcus aureus is a human and animal pathogen as well as a commensal bacterium. It can be a causative agent of severe, life-threatening infections with high mortality, e.g., toxic shock syndrome, septic shock, and multi-organ failure. S. aureus strains secrete a number of toxins. Exotoxins/enterotoxins are considered important in the pathogenesis of the above-mentioned conditions. Exotoxins, e.g., superantigen toxins, cause uncontrolled and polyclonal T cell activation and unregulated activation of inflammatory cytokines. Here we show the importance of genomic analysis of infectious strains in order to identify disease-causing exotoxins. Further, we show through functional analysis of superantigenic properties of staphylococcal exotoxins that even very small amounts of a putative superantigenic contaminant can have a significant mitogenic effect. The results show expression and production of two distinct staphylococcal exotoxins, SEC and SEL, in several strains from clinical isolates. Antibodies against both toxins are required to neutralise the superantigenic activity of staphylococcal supernatants and purified staphylococcal toxins.

Staphylococcal superantigen toxins lead to a devastating cytokine storm resulting in shock and multi-organ failure. We have previously assessed the safety and immunogenicity of a recombinant toxic shock syndrome toxin 1 variant vaccine (rTSST-1v) in clinical trials (NCT02971670 and NCT02340338). The current study assessed neutralizing antibody titers after repeated vaccination with escalating doses of rTSST-1v. At study entry, 23 out of 34 subjects (67.6%) had neutralizing antibody titers inhibiting T cell activation as determined by 3 H-thymidine incorporation at a serum dilution of ≤1:100 with similar figures for inhibition of IL-2 activation (19 of 34 subjects, 55.9%) as assessed by quantitative PCR. After the first vaccination, numbers of subjects with neutralization titers inhibiting T cell activation (61.7% ≥ 1:1000) and inhibiting IL-2 gene induction (88.2% ≥ 1:1000) increased. The immune response was augmented after the second vaccination (inhibiting T cell activation: 78.8% ≥ 1:1000; inhibiting IL-2 induction: 93.9% ≥ 1:1000) corroborated with a third immunization months later in a small subgroup of subjects. Assessment of IFNγ, TNFα and IL-6 inhibition revealed similar results, whereas neutralization titers did not change in placebo participants. Antibody titer studies show that vaccination with rTSST-1v in subjects with no/low neutralizing antibodies can rapidly induce high titer neutralizing antibodies persisting over months.

Staphylococcal enterotoxins are the most common cause of foodborne intoxications (staphylococcal food poisoning) and cause a wide range of diseases. With at least six variants staphylococcal enterotoxin C (SEC) stands out as particularly diverse amongst the 25 known staphylococcal enterotoxins. Some variants present unique and even host-specific features. Here, we review the role of SEC in human and animal health with a particular focus on its role as a causative agent for foodborne intoxications. We highlight structural features unique to SEC and its variants, particularly, the emetic and superantigen activity, as well as the roles of SEC in mastitis and in dairy products. Information about the genetic organization as well as regulatory mechanisms including the accessory gene regulator and food-related stressors are provided.

Staphylococcal toxic shock syndrome is a potentially lethal illness attributed to superantigens produced by Staphylococcus aureus , in particular toxic shock syndrome toxin 1 (TSST-1), but staphylococcal enterotoxins (SEs) are also implicated. The genes encoding these important toxins are carried on mobile genetic elements, and the regulatory networks controlling expression of these toxins remain relatively unexplored. We show here that the highly conserved ClpXP protease stimulates transcription of tst (TSST-1), sec (SEC), and sed (SED) genes in the prototypical strains, SA564 and RN4282. In the wild-type cells, the post-exponential upregulation of toxin gene transcription was proposed to occur via RNAIII-mediated downregulation of the Rot repressor. Contradictive to this model, we showed that the post-exponential induction of tst , sed, and sec transcription did not occur in cells devoid of ClpXP activity, despite the Rot level being diminished. To identify transcriptional regulators with a changed expression in cells devoid of ClpXP activity, RNA sequencing was performed. The RNAseq analysis revealed a number of global virulence regulators that might act downstream of ClpXP, to control expression of tst and other virulence genes. Collectively, the results extend our understanding of the complex transcriptional regulation of the tst, sed, and sec genes., Competing Interests: The authors declare no conflict of interest.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes substantial skin and soft tissue infections annually in the United States and expresses numerous virulence factors, including a family of toxins known as the staphylococcal superantigen-like (SSL) proteins. Many of the SSL protein structures have been determined and implicated in immune system avoidance, but the full scope that these proteins play in different infection contexts remains unknown and continues to warrant investigation. Analysis of ssl gene regulation may provide valuable information related to the function of these proteins. To determine the transcriptional regulation of the ssl1 gene of CA-MRSA strain MW2, an ssl1 promoter::lux fusion was constructed and transformed into S. aureus strains RN6390 and Newman. Resulting strains were grown in a defined minimal medium (DSM) broth and nutrient-rich brain-heart infusion (BHI) broth and expression was determined by luminescence. Transcription of ssl1 was up-regulated and occurred earlier during growth in DSM broth compared to BHI broth suggesting expression is regulated by nutrient availability. RN6390 and Newman strains containing the ssl1::lux fusion were also used to analyze regulation in vivo using a mouse abscess model of infection. A marked increase in ssl1 transcription occurred early during infection, suggesting SSL1 is important during early stages of infection, perhaps to avoid the immune system.

Staphylococcus aureus is a major cause of infections. Toxic shock syndrome toxin (TSST-1) and Panton-Valentine leukocidin (PVL) are associated with severe clinical syndromes. S. aureus colonizing isolates recovered from healthcare workers and patients in the intensive care unit (ICU) of a university hospital comprising Group A were compared with those from adult non-ICU carriers (Group B). mecA , lukS/lukF-PV (Panton-Valentine leukocidin, PVL), and tst (toxic shock syndrome toxin) gene carriage was detected by PCR. Clones were identified in all methicillin-resistant S. aureus (MRSA) and toxin-positive methicillin-susceptible strains (MSSA) by pulsed-field gel electrophoresis (PFGE), agr groups, and multi locus sequencing typing (MLST). Group A included 90 S. aureus isolates, whereas Group B 53. PVL was more frequently found among MRSA vs. MSSA ( p < 0.001) and in strains of Group B as compared to Group A ( p < 0.001), consistent with the spread of ST80-IV. Higher incidence of tst gene carriage was identified among MSSA vs. MRSA ( P 0.005) belonging mainly to ST30, and Group A vs. Group B ( P 0.002). The wide dissemination of ST80-IV mainly in the community is responsible for a high percentage of PVL-positive MRSA, while silent spread of tst -positive S. aureus clones among ICU patients and personnel poses a threat of hospital transmission and possible severe infections., Competing Interests: E.D. has received financial support from the National Staphylococcal Reference Laboratory. All other authors declare no conflicts of interest.

Asymptomatic carriers of toxigenic Staphylococcus aureus are potential source of diseases, including food poisoning. Toxigenic potential and genetic traits of colonizing S. aureus were investigated for 563 healthy food handlers in Myanmar. Carriage of S. aureus was found in 110 individuals (19.5%), and a total of 144 S. aureus isolates were recovered from nasal cavities (110 isolates) and hands (34 isolates). Panton-Valentine leucocidin genes ( pvl ) were detected in 18 isolates (12.5%), among which 11 isolates were classified into coa -VIa, agr type III, and ST1930 (CC96) that had been also detected in pvl -positive clinical isolates in Myanmar. A pvl -positive, ST2250 nasal isolate was identified as S. argenteus , a novel coagulase-positive staphylococcus species. Toxic shock syndrome toxin-1 (TSST-1) gene was detected in five pvl -negative isolates. All of the 144 isolates harbored at least one of the 21 enterotoxin(-like) gene(s). The most prevalent enterotoxin(-like) gene was selw (98%), followed by selx (97%), sei (28%), sely (28%), sem (26%), sel (24%), and sea and sec (22% each). Considerable genetic diversity with five groups was detected for selw . The present study revealed the relatively high rate of pvl , as well as the wide distribution of enterotoxin(-like) genes among colonizing S. aureus in Myanmar., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Some Staphylococcus aureus isolates produced toxic shock syndrome toxin-1 (TSST-1) which is a pyrogenic toxin superantigen (PTSAg). The toxin activates a large fraction of peripheral blood T lymphocytes causing the cells to proliferate and release massive amounts of pro-inflammatory cytokines leading to a life-threatening multisystem disorder: toxic shock syndrome (TSS). PTSAg-mediated-T cell stimulation circumvents the conventional antigenic peptide presentation to T cell receptor (TCR) by the antigen-presenting cell (APC). Instead, intact PTSAg binds directly to MHC-II molecule outside peptide binding cleft and simultaneously cross-links TCR-Vβ region. Currently, there is neither specific TSS treatment nor drug that directly inactivates TSST-1. In this study, human single chain antibodies (HuscFvs) that bound to and neutralized bioactivities of the TSST-1 were generated using phage display technology. Three E. coli clones transfected with TSST-1-bound phages fished-out from the human scFv library using recombinant TSST-1 as bait expressed TSST-1-bound-HuscFvs that inhibited the TSST-1-mediated T cell activation and pro-inflammatory cytokine gene expressions and productions.Computerized simulation, verified by mutations of the residues of HuscFv complementarity determining regions (CDRs),predicted to involve in target binding indicated that the HuscFvs formed interface contact with the toxin residues important for immunopathogenesis. The HuscFvs have high potential for future therapeutic application.

Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SE l N, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SE l N led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSE l N led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens., Competing Interests: The authors declare no conflict of interest.

Toxic shock syndrome (TSS) results from the host's overwhelming inflammatory response and cytokine storm mainly due to superantigens (SAgs). There is no effective specific therapy. Application of immunoglobulins has been shown to improve the outcome of the disease and to neutralize SAgs both in vivo and in vitro. However, in most experiments that have been performed, antiserum was either pre-incubated with SAg, or both were applied simultaneously. To mirror more closely the clinical situation, we applied a multiple dose (over five days) lethal challenge in a rabbit model. Treatment with toxic shock syndrome toxin 1 (TSST-1) neutralizing antibody was fully protective, even when administered late in the course of the challenge. Kinetic studies on the effect of superantigen toxins are scarce. We performed in vitro kinetic studies by neutralizing the toxin with antibodies at well-defined time points. T-cell activation was determined by assessing T-cell proliferation (3H-thymidine incorporation), determination of IL-2 release in the cell supernatant (ELISA), and IL-2 gene activation (real-time PCR (RT-PCR)). Here we show that T-cell activation occurs continuously. The application of TSST-1 neutralizing antiserum reduced IL-2 and TNFα release into the cell supernatant, even if added at later time points. Interference with the prolonged stimulation of proinflammatory cytokines is likely to be in vivo relevant, as postexposure treatment protected rabbits against the multiple dose lethal SAg challenge. Our results shed new light on the treatment of TSS by specific antibodies even at late stages of exposure.

Staphylococcal superantigens (sAgs), such as toxic shock syndrome toxin 1 (TSST-1), induce massive cytokine production, which may result in toxic shock syndrome (TSS) and sepsis. Recently, we reported that in vitro studies in human peripheral blood mononuclear cells (PBMC) do not reflect the immunological situation of the host, because after exposure to superantigens (sAgs) in vivo, mononuclear cells (MNC) leave the circulation and migrate to organs, e.g., the spleen, liver and lung. Our experimental model of choice is the rabbit because it is comparable to humans in its sensitivity to sAg. T cell activation has been assessed by lymphocyte proliferation and IL-2 gene expression after in vivo challenge with TSST-1 and the mutant antigens; expression of the genes of proinflammatory cytokines were taken as indicators for the inflammatory reaction after the combined treatment with TSST-1 and LPS. The question as to whether the biological activities of TSST-1, e.g., lymphocyte extravasation, toxicity and increased sensitivity to LPS, are mediated by T cell activation or activation by MHC II-only, are unresolved and results are contradictory. We have addressed this question by studying these reactions in vivo, with two TSST-1 mutants: one mutated at the MHC binding site (G31R) with reduced MHC binding with residual activity still present, and the other at the T cell binding site (H135A) with no residual function detectable. Here, we report that the mutant G31R induced all the biological effects of the wild type sAg, while the mutant with non-functional TCR binding did not retain any of the toxic effects, proving the pivotal role of T cells in this system.