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Background The Assessment of SpondyloArthritis international Society Health Index (ASAS HI) is a novel questionnaire of global functioning for patients with axial spondyloarthritis (SpA).Objective The objective was to assess the construct validity, discriminatory ability and responsiveness of ASAS HI in relation to patient-reported outcome measures (PROMs), MRI and radiography.Methods Data from two longitudinal studies with tumour necrosis factor inhibitor (TNFi) initiation (novel MRI And biomarkers in Golimumab-treated patients with axial spondyloarthritis (MANGO): n=45) respectively tapering (Dose adjustment of Biological treatment in patients with SpA (DOBIS): n=106) were used. Analyses included a wide panel of PROMs, MRI and radiography scores of the spine and sacroiliac joints (SIJs).Results In the MANGO study, 30 (68%) patients were clinical responders at week 16. In the DOBIS study, 105 (99%) patients flared after mean (SD; min–max) 31 (17; 2.7–81) weeks. After initiation of TNF inhibitor in MANGO, ASAS HI significantly decreased from baseline to week 4, 16 and 52. In DOBIS, ASAS significantly increased from baseline to the flare visit and significantly decreased from the flare visit to week 96. In multivariate regression models, ASAS HI was independently associated with Spondyloarthritis Research Consortium of Canada MRI SIJ Inflammation score, Canada-Denmark MRI Spine Inflammation score, EuroQol, Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Disease Activity Index and Patient Global. Patients stratified according to ASAS HI health status groups (good, moderate, poor) at baseline and change categories (absolute and percentage change) from baseline to week 16/flare showed good discriminatory ability for almost all outcome variables (p≤0.001). ASAS HI had a large responsiveness in MANGO (standardised response mean (SRM)=−1.3, effect size (ES)=−1.7) and moderate responsiveness in DOBIS (SRM=0.7, ES=0.6).Conclusion ASAS HI showed good construct validity, discriminatory ability and responsiveness.Trial registration number ClinicalTrials.gov: NCT02011386.

Abstract Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel’s cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel’s cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

Objective: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. Methods: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. Results: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. Conclusions: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.

Abstract Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor‐mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C‐type lectin‐like receptor 2 and combining all thrombin‐collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor‐induced PEVs. Our findings underscore that PEVs are tunable through receptor‐mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.

Abstract The genetic architecture of the QT interval, defined as the period from onset of depolarisation to completion of repolarisation of the ventricular myocardium, is incompletely understood. Only a minor part of the QT interval variation in the general population has been linked to autosomal variant loci. Altered X chromosome dosage in humans, as seen in sex chromosome aneuploidies such as Turner syndrome (TS) and Klinefelter syndrome (KS), is associated with altered QTc interval (heart rate corrected QT), indicating that genes, located in the pseudoautosomal region 1 of the X and Y chromosomes may contribute to QT interval variation. We investigate the dosage effect of the pseudoautosomal gene SLC25A6, encoding the membrane ADP/ATP translocase 3 in the inner mitochondrial membrane, on QTc interval duration. To this end we used human participants and in vivo zebrafish models. Analyses in humans, based on 44 patients with KS, 44 patients with TS, 59 male and 22 females, revealed a significant negative correlation between SLC25A6 expression level and QTc interval duration. Similarly, downregulation of slc25a6 in zebrafish increased QTc interval duration with pharmacological inhibition of KATP channels restoring the systolic duration, whereas overexpression of SLC25A6 shortened QTc, which was normalized by pharmacological activation of KATP channels. Our study demonstrate an inverse relationship between SLC25A6 dosage and QTc interval indicating that SLC25A6 contributes to QT interval variation.

Summary: Motor neuron (MN) development and nerve regeneration requires orchestrated action of a vast number of molecules. Here, we identify SorCS2 as a progranulin (PGRN) receptor that is required for MN diversification and axon outgrowth in zebrafish and mice. In zebrafish, SorCS2 knockdown also affects neuromuscular junction morphology and fish motility. In mice, SorCS2 and PGRN are co-expressed by newborn MNs from embryonic day 9.5 until adulthood. Using cell-fate tracing and nerve segmentation, we find that SorCS2 deficiency perturbs cell-fate decisions of brachial MNs accompanied by innervation deficits of posterior nerves. Additionally, adult SorCS2 knockout mice display slower motor nerve regeneration. Interestingly, primitive macrophages express high levels of PGRN, and their interaction with SorCS2-positive motor axon is required during axon pathfinding. We further show that SorCS2 binds PGRN to control its secretion, signaling, and conversion into granulins. We propose that PGRN-SorCS2 signaling controls MN development and regeneration in vertebrates.

Abstract Background Dual-energy CT (DECT) can acknowledge differences in tissue compositions and can colour-code tissues with specific features including monosodium urate (MSU) crystals. However, when evaluating gout patients, DECT frequently colour-codes material not truly representing MSU crystals and this might lead to misinterpretations. The characteristics of and variations in properties of colour-coded DECT lesions in gout patients have not yet been systematically investigated. The objective of this study was to evaluate the properties and locations of colour-coded DECT lesions in gout patients. Methods DECT of the hands, knees and feet were performed in patients with suspected gout using factory default gout settings, and colour-coded DECT lesions were registered. For each lesion, properties [mean density (mean of Hounsfield Units (HU) at 80 kV and Sn150kV), mean DECT ratio and size] and location were determined. Subgroup analysis was performed post hoc evaluating differences in locations of lesions when divided into definite MSU depositions and possibly other lesions. Results In total, 4033 lesions were registered in 27 patients (23 gout patients, 3918 lesions; 4 non-gout patients, 115 lesions). In gout patients, lesions had a median density of 160.6 HU and median size of 6 voxels, and DECT ratios showed an approximated normal distribution (mean 1.06, SD 0.10), but with a right heavy tail consistent with the presence of smaller amounts of high effective atomic number lesions (e.g. calcium-containing lesions). The most common locations of lesions were 1st metatarsophalangeal (MTP1), knee and midtarsal joints along with quadriceps and patella tendons. Subgroup analyses showed that definite MSU depositions (large volume, low DECT ratio, high density) had a similar distribution pattern, whereas possible calcium-containing material (high DECT ratio) and non-gout MSU-imitating lesions (properties as definite MSU depositions in non-gout patients) were primarily found in some larger joints (knee, midtarsal and talocrural) and tendons (Achilles and quadriceps). MTP1 joints and patella tendons showed only definite MSU depositions. Conclusion Colour-coded DECT lesions in gout patients showed heterogeneity in properties and distribution. MTP1 joints and patella tendons exclusively showed definite MSU depositions. Hence, a sole focus on these regions in the evaluation of gout patients may improve the specificity of DECT scans.

The wide diversity of cortical inhibitory neuron types populating the cortex allows the assembly of diverse microcircuits and endows these circuits with different computational properties. Thus, characterizing neuronal diversity is fundamental to describe the building blocks of cortical microcircuits and probe their function. To this purpose, the mouse has emerged as a powerful tool to genetically label and manipulate specific inhibitory cell-types in the mammalian brain. Among these cell-types, the parvalbumin-expressing interneuron type (PV-INs) is perhaps the most characterized. Several mouse lines have been generated to target PV-INs. Among these mouse lines, the PV-IRES-Cre lines is the most widely used and demonstrated a high specificity and efficiency in targeting PV-INs in different cortical areas. However, a characterization of the performance across cortical regions is still missing. Here we show that the PV-IRES-Cre mouse line labels only a fraction of PV immunoreactive neurons in perirhinal cortex and other association areas. Our results point to a yet uncharacterized diversity within the PV-INs and emphasize the need to characterize these tools in specific cortical areas.

Human patients carrying PAPP‐A2 inactivating mutations have low bone mineral density. The underlying mechanisms for this reduced calcification are poorly understood. Using a zebrafish model, we report that Papp-aa regulates bone calcification by promoting Ca2+-transporting epithelial cell (ionocyte) quiescence-proliferation transition. Ionocytes, which are normally quiescent, re-enter the cell cycle under low [Ca2+] stress. Genetic deletion of Papp-aa, but not the closely related Papp-ab, abolished ionocyte proliferation and reduced calcified bone mass. Loss of Papp-aa expression or activity resulted in diminished IGF1 receptor-Akt-Tor signaling in ionocytes. Under low Ca2+ stress, Papp-aa cleaved Igfbp5a. Under normal conditions, however, Papp-aa proteinase activity was suppressed and IGFs were sequestered in the IGF/Igfbp complex. Pharmacological disruption of the IGF/Igfbp complex or adding free IGF1 activated IGF signaling and promoted ionocyte proliferation. These findings suggest that Papp-aa-mediated local Igfbp5a cleavage functions as a [Ca2+]-regulated molecular switch linking IGF signaling to bone calcification by stimulating epithelial cell quiescence-proliferation transition under low Ca2+ stress.

Mutations in potassium and calcium channel genes have been associated with cardiac arrhythmias. Here, Jensen et al. show that an anion transporter chloride-bicarbonate exchanger AE3 is also responsible for the genetically-induced mechanism of cardiac arrhythmia, suggesting new therapeutic targets for this disease

Formation of functional myelin sheaths within the central nervous system depends on expression of myelin basic protein (MBP). Following process extension and wrapping around axonal segments, this highly basic protein is required for compaction of the multi-layered membrane sheath produced by oligodendrocytes. MBP is hypothesized to be targeted to the membrane sheath by mRNA transport and local translation, which ensures that its expression is temporally and spatially restricted. The mechanistic details of how this might be regulated are still largely unknown, in particular because a model system that allows this process to be studied in vivo is lacking. We here show that the expression of the zebrafish MBP orthologs, mbpa and mbpb, is developmentally regulated, and that expression of specific mbpa isoforms is restricted to the peripheral nervous system. By analysis of transgenic zebrafish, which express a fluorescent reporter protein specifically in myelinating oligodendrocytes, we demonstrate that both mbpa and mbpb include a 3’UTR sequence, by which mRNA transport and translation is regulated in vivo. Further functional analysis suggests that: (1) the 3’UTRs delay the onset of protein expression; and that (2) several regulatory elements contribute to targeting of the mbp mRNA to the myelin sheath. Finally, we show that a pharmacological compound known to enhance neuronal activity stimulates the translation of Mbp in zebrafish in a 3’UTR-dependent manner. A similar effect was obtained following stimulation with a TrkB receptor agonist, and cell-based assays further confirmed that the receptor ligand, BDNF, in combination with other signals reversed the inhibitory effect of the 3’UTR on translation.

Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

Staphylococcus aureus is a predominant cause of post-operative surgical site infections and persistent bacteremia. Here, we describe a patient who experienced three episodes of S. aureus infection over a period of 4 months following a total knee arthroplasty. The initial bloodstream isolate (SAB-0429) was a clonal complex 5 (CC5) and methicillin-resistant S. aureus (MRSA), whereas two subsequent isolates (SAB-0485 and SAB-0495) were CC5 isolates but methicillin-sensitive S. aureus . The two latter isolates harbored a plasmid encoding three superantigen genes that were not present in the primary MRSA isolate. SAB-0485 and SAB-0495 both expressed the plasmid-encoded staphylococcal enterotoxin R exotoxin and demonstrated increased superantigen activity compared with SAB-0429. Compared to SAB-0429, the latter isolates also demonstrated an increased bacterial burden in a mouse bacteremia model that was dependent on increased interferon-γ production. Curing of the plasmid from SAB-0485 reduced this virulence phenotype. These findings suggest that the superantigen exotoxins may provide a selective advantage in chronic post-surgical infections.IMPORTANCEIn this study, we investigated bacterial isolates from a patient who experienced three recurrent S. aureus infections over a 4 month period following total knee arthroplasty. Genomic and phenotypic characterization of these isolates revealed that they all belonged to clonal complex 5, yet the latter two strains contained an additional plasmid encoding superantigen exotoxins. Subsequent experimental infection experiments in mice demonstrated that the plasmid-encoded superantigens exacerbated bacteremia by promoting liver abscess formation. These experiments suggest that despite appropriate antibiotic therapy, bacterial superantigens may be able to promote persistent infection following post-surgery.

Toxic shock syndrome toxin-1 (TSST-1) is a superantigen produced by Staphylococcus aureus and is the determinant of menstrual toxic shock syndrome (mTSS); however, the impact of TSST-1 on the vaginal environment beyond mTSS is not understood. Herein, we assessed how TSST-1 affects vaginal colonization by S. aureus , host inflammatory responses, and changes in microbial communities within the murine vagina. We demonstrated that TSST-1 induced a CD8 + T-cell-dependent inflammatory response in 24 h that correlated with S. aureus persistence within the vaginal tract. This increase was due to superantigen-dependent T-cell activation that triggered a change in microbial composition within the vaginal tract. Altogether, this study demonstrates that within the vaginal tract, TSST-1 modulates the vaginal microbiota to favor the survival of S. aureus in the absence of mTSS.IMPORTANCEToxic shock syndrome toxin-1 (TSST-1) is a superantigen toxin produced from Staphylococcus aureus that causes the menstrual form of toxic shock syndrome. This research demonstrates that TSST-1 also has a wider function within the vaginal tract than previously expected. We show that TSST-1, by activating CD8 + T cells, induces an inflammatory environment that modifies the vaginal microbiota to favor colonization by S. aureus . These are important findings as S. aureus can colonize the human vaginal tract efficiently and subsequently trigger dysbiosis within the microbial communities leading to several adverse outcomes such as decreased fertility, increased risks for sexually transmitted diseases, and issues related to pregnancy and birth.

Streptococcus pyogenes is a human-specific pathogen that commonly colonizes the upper respiratory tract and skin, causing a wide variety of diseases ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome. S. pyogenes has a repertoire of secreted virulence factors that promote infection and evasion of the host immune system including the cytolysins streptolysin O (SLO) and streptolysin S (SLS). S. pyogenes does not naturally infect the upper respiratory tract of mice although mice transgenic for MHC class II human leukocyte antigens (HLA) become highly susceptible. Here we used HLA-transgenic mice to assess the role of both SLO and SLS during both nasopharyngeal and skin infection. Using S. pyogenes MGAS8232 as a model strain, we found that an SLS-deficient strain exhibited a 100-fold reduction in bacterial recovery from the nasopharynx and a 10-fold reduction in bacterial burden in the skin, whereas an SLO-deficient strain did not exhibit any infection defects in these models. Furthermore, depletion of neutrophils significantly restored the bacterial burden of the SLS-deficient bacteria in skin, but not in the nasopharynx. In mice nasally infected with the wildtype S. pyogenes, there was a marked change in localization of the tight junction protein ZO-1 at the site of infection, demonstrating damage to the nasal epithelia that was absent in mice infected with the SLS-deficient strain. Overall, we conclude that SLS is required for the establishment of nasopharyngeal infection and skin infection in HLA-transgenic mice by S. pyogenes MGAS8232 and provide evidence that SLS contributes to nasopharyngeal infection through the localized destruction of nasal epithelia., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shannon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Staphylococcus aureus is a foremost bacterial pathogen responsible for a vast array of human diseases. Staphylococcal superantigens (SAgs) constitute a family of exotoxins from S. aureus that bind directly to major histocompatibility complex (MHC) class II and T cell receptors to drive extensive T cell activation and cytokine release. Although these toxins have been implicated in serious disease, including toxic shock syndrome, the specific pathological mechanisms remain unclear. Herein, we aimed to elucidate how SAgs contribute to pathogenesis during bloodstream infections and utilized transgenic mice encoding human MHC class II to render mice susceptible to SAg activity. We demonstrate that SAgs contribute to S. aureus bacteremia by massively increasing bacterial burden in the liver, and this was mediated by CD4 + T cells that produced interferon gamma (IFN-γ) to high levels in a SAg-dependent manner. Bacterial burdens were reduced by blocking IFN-γ, phenocopying SAg-deletion mutant strains, and inhibiting a proinflammatory response. Infection kinetics and flow cytometry analyses suggested that this was a macrophage-driven mechanism, which was confirmed through macrophage-depletion experiments. Experiments in human cells demonstrated that excessive IFN-γ allowed S. aureus to replicate efficiently within macrophages. This indicates that SAgs promote bacterial survival by manipulating the immune response to inhibit effective clearing of S. aureus Altogether, this work implicates SAg toxins as critical therapeutic targets for preventing persistent or severe S. aureus disease., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins.

Background: Injection drug use-associated endocarditis (IDUaIE) incidence in Ontario has recently been associated with hydromorphone prescribing rates. Staphylococcus aureus causes the majority of cases of IDUaIE in Ontario and across North America. Hydromorphone controlled-release (Hydromorphone-CR) requires a complex technique for injection and therefore provides multiple opportunities for contamination. Hydromorphone-CR contains several excipients, which could enhance staphylococcal survival and increase risk of contaminating the injectate., Methods: Used injection drug preparation equipment (cookers/filters) was collected from persons who inject drugs (PWID), rinsed with water, and plated on Mannitol salt agar. Bacterial isolates from bacteremic PWID were used to assess the survival of S. aureus and Streptococcus pyogenes on cookers/filters with Hydromorphone-CR, hydromorphone immediate-release (Hydromorphone-IR) or oxycodone controlled-release (Oxycodone-CR). The solutions spiked with S. aureus were heated and the remaining viable bacteria enumerated., Results: S. aureus was detected in 12/87 (14%, 95%CI 8-23%) cookers/filters samples used for injection of Hydromorphone-CR. Hydromorphone-CR was the only opioid associated with greater survival of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) on cookers/filters when compared to sterile water vehicle control. There was a ~2 log reduction in the number of S. aureus that survived when cookers/filters were heated., Conclusion: 14% of all cookers/filters used in the preparation of Hydromorphone-CR were contaminated with S. aureus. Hydromorphone-CR prolongs the survival of MRSA and MSSA in cookers/filters. Heating cookers/filters may be a harm-reduction strategy., Competing Interests: The authors have declared that no competing interests exist.

Streptococcus pyogenes is a human-specific and globally prominent bacterial pathogen that despite causing numerous human infections, this bacterium is normally found in an asymptomatic carrier state. This review provides an overview of both bacterial and human factors that likely play an important role in nasopharyngeal colonization and pharyngitis, as well as the development of acute rheumatic fever and rheumatic heart disease. Here we highlight a recently described role for bacterial superantigens in promoting acute nasopharyngeal infection, and discuss how these immune system activating toxins could be crucial to initiate the autoimmune process in rheumatic heart disease., (Copyright © 2018. Published by Elsevier B.V.)

The globally prominent pathogen Streptococcus pyogenes secretes potent immunomodulatory proteins known as superantigens (SAgs), which engage lateral surfaces of major histocompatibility class II molecules and T-cell receptor (TCR) β-chain variable domains (Vβs). These interactions result in the activation of numerous Vβ-specific T cells, which is the defining activity of a SAg. Although streptococcal SAgs are known virulence factors in scarlet fever and toxic shock syndrome, mechanisms by how SAgs contribute to the life cycle of S. pyogenes remain poorly understood. Herein, we demonstrate that passive immunization against the Vβ8-targeting SAg streptococcal pyrogenic exotoxin A (SpeA), or active immunization with either wild-type or a nonfunctional SpeA mutant, protects mice from nasopharyngeal infection; however, only passive immunization, or vaccination with inactive SpeA, resulted in high-titer SpeA-specific antibodies in vivo. Mice vaccinated with wild-type SpeA rendered Vβ8 + T cells poorly responsive, which prevented infection. This phenotype was reproduced with staphylococcal enterotoxin B, a heterologous SAg that also targets Vβ8 + T cells, and rendered mice resistant to infection. Furthermore, antibody-mediated depletion of T cells prevented nasopharyngeal infection by S. pyogenes , but not by Streptococcus pneumoniae , a bacterium that does not produce SAgs. Remarkably, these observations suggest that S. pyogenes uses SAgs to manipulate Vβ-specific T cells to establish nasopharyngeal infection., Competing Interests: The authors declare no conflict of interest.

Unlabelled: Toxic shock syndrome toxin 1 (TSST-1) is a Staphylococcus aureus superantigen that has been implicated in both menstrual and nonmenstrual toxic shock syndrome (TSS). Despite the important role of TSST-1 in severe human disease, a comprehensive understanding of staphylococcal regulatory factors that control TSST-1 expression remains incomplete. The S. aureus exotoxin expression (Sae) operon contains a well-characterized two-component system that regulates a number of important exotoxins in S. aureus, although regulation of TSST-1 by the Sae system has not been investigated. We generated a defined deletion mutant of the Sae histidine kinase sensor (saeS) in the prototypic menstrual TSS strain S. aureus MN8. Mutation of saeS resulted in a complete loss of TSST-1 expression. Using both luciferase reporter experiments and quantitative real-time PCR, we demonstrate that the Sae system is an important transcriptional activator of TSST-1 expression. Recombinant SaeR was able to bind directly to the tst promoter to a region containing two SaeR consensus binding sites. Although the stand-alone SarA transcriptional regulator has been shown to be both a positive and a negative regulator of TSST-1, deletion of sarA in S. aureus MN8 resulted in a dramatic overexpression of TSST-1. As expected, mutation of agr also reduced TSST-1 expression, but this phenotype appeared to be independent of Sae. A double mutation of saeS and sarA resulted in the loss of TSST-1 expression. This work indicates that the Sae system is a dominant and direct transcriptional activator that is required for expression of TSST-1., Importance: The TSST-1 superantigen is an exotoxin, produced by some strains of S. aureus, that has a clear role in both menstrual and nonmenstrual TSS. Although the well-characterized agr quorum sensing system is a known positive regulator of TSST-1, the molecular mechanisms that directly control TSST-1 expression are only partially understood. Our studies demonstrate that the Sae two-component regulatory system is a positive transcriptional regulator that binds directly to the TSST-1 promoter, and furthermore, our data suggest that Sae is required for expression of TSST-1. This work highlights how major regulatory circuits can converge to fine-tune exotoxin expression and suggests that the Sae regulatory system may be an important target for antivirulence strategies., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Streptococcus pyogenes is a globally prominent human-specific pathogen that is responsible for an enormous burden of infectious disease. Despite intensive experimental efforts to understand the molecular correlates that contribute to invasive infections, there has been less focus on S. pyogenes carriage and local infection of the nasopharynx. This chapter describes an acute nasopharyngeal infection model in mice that is utilized in our laboratory to study the role of superantigen toxins in the biology of S. pyogenes. We also describe a method to detect superantigen-specific T cell activation in vivo.

Superantigens (SAgs) are potent microbial toxins that function to activate large numbers of T cells in a T cell receptor (TCR) Vβ-specific manner, resulting in excessive immune system activation. Staphylococcus aureus possesses a large repertoire of distinct SAgs, and in the context of host-pathogen interactions, staphylococcal SAg research has focused primarily on the role of these toxins in severe and invasive diseases. However, the contribution of SAgs to colonization by S. aureus remains unclear. We developed a two-week nasal colonization model using SAg-sensitive transgenic mice expressing HLA-DR4, and evaluated the role of SAgs using two well-studied stains of S. aureus. S. aureus Newman produces relatively low levels of staphylococcal enterotoxin A (SEA), and although we did not detect significant TCR-Vβ specific changes during wild-type S. aureus Newman colonization, S. aureus Newman Δsea established transiently higher bacterial loads in the nose. S. aureus COL produces relatively high levels of staphylococcal enterotoxin B (SEB), and colonization with wild-type S. aureus COL resulted in clear Vβ8-specific T cell skewing responses. S. aureus COL Δseb established consistently higher bacterial loads in the nose. These data suggest that staphylococcal SAgs may be involved in regulating bacterial densities during nasal colonization.

Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as 'trademark' virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC -II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms.

Background: Fournier's gangrene is a rare necrotizing soft tissue infection of the scrotum and penis. We report, to our knowledge, the first case of Fournier's gangrene caused by Streptococcus dysgalactiae subsp. equisimilis (SDSE), a strain of pyogenic β-hemolytic streptococci that is increasingly being recognized as an important human pathogen., Case Presentation: We describe a healthy 59 year-old Caucasian male who presented to the emergency department with Fournier's gangrene of the penis and scrotum, with extension to the anterior abdominal wall. He underwent urgent surgical debridement of his scrotum, penis, and anterior abdomen. Swabs from the scrotum grew Gram-positive cocci, which were initially identified as Streptococcus anginosus group by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). However, polymerase chain reaction (PCR) amplification and sequencing of the 16S rRNA gene identified the isolate as Streptococcus dysgalatiae subspecies equisimilis (SDSE). The incidences of invasive S. anginosus group and SDSE infections at the London Health Sciences Centre, a tertiary-care institution in southwestern Ontario, were determined between August 1, 2011 and August 31, 2012, revealing a slightly lower rate of SDSE (3.2 cases per 100,000 population) than other studies., Conclusions: This case highlights a unique disease manifestation of the emerging human pathogen Streptococcus dysgalatiae subspecies equisimilis that has not been previously reported. This case also underscores the limitations of MALDI-TOF MS in differentiating between closely-related streptococcal species which may have different pathogenic profiles.

Superantigens (SAgs) are microbial toxins defined by their ability to activate T lymphocytes in a T cell receptor (TCR) β-chain variable domain (Vβ)-specific manner. Although existing structural information indicates that diverse bacterial SAgs all uniformly engage the Vβ second complementarity determining region (CDR2β) loop, the molecular rules that dictate SAg-mediated T cell activation and Vβ specificity are not fully understood. Herein we report the crystal structure of human Vβ2.1 (hVβ2.1) in complex with the toxic shock syndrome toxin-1 (TSST-1) SAg, and mutagenesis of hVβ2.1 indicates that the non-canonical length of CDR2β is a critical determinant for recognition by TSST-1 as well as the distantly related SAg streptococcal pyrogenic exotoxin C. Frame work (FR) region 3 is uniquely critical for TSST-1 function explaining the fine Vβ-specificity exhibited by this SAg. Furthermore, domain swapping experiments with SAgs, which use distinct domains to engage both CDR2β and FR3/4β revealed that the CDR2β contacts dictate T lymphocyte Vβ-specificity. These findings demonstrate that the TCR CDR2β loop is the critical determinant for functional recognition and Vβ-specificity by diverse bacterial SAgs.

Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome.

Staphylococcal superantigens are pyrogenic exotoxins that cause massive T cell activation leading to toxic shock syndrome and death. Despite the strong adaptive immune response induced by these toxins, infections by superantigen-producing staphylococci are very common clinical events. We hypothesized that this may be partly a result of staphylococcal strains having developed strategies that downregulate the T cell response to these toxins. Here we show that the human interleukin-2 response to staphylococcal superantigens is inhibited by the simultaneous presence of bacteria. Such a downregulatory effect is the result of peptidoglycan-embedded molecules binding to Toll-like receptor 2 and inducing interleukin-10 production and apoptosis of antigen-presenting cells. We corroborated these findings in vivo by showing substantial prevention of mortality after simultaneous administration of staphylococcal enterotoxin B with either heat-killed staphylococci or Staphylococcus aureus peptidoglycan in mouse models of superantigen-induced toxic shock syndrome.

Superantigens (SAgs) are microbial toxins that bind to both TCR beta-chain variable domains (Vbetas) and MHC class II molecules, resulting in the activation of T cells in a Vbeta-specific manner. It is now well established that different isoforms of MHC II molecules can play a significant role in the immune response to bacterial SAgs. In this work, using directed mutational studies in conjunction with functional analyses, we provide a complete functional map of the low-affinity MHC II alpha-chain binding interface of the SAg streptococcal pyrogenic exotoxin C (SpeC) and identify a functional epitope in the beta-barrel domain that is required for the activation of T cells. Using cell lines that exclusively express individual MHC II isoforms, our studies provide a molecular basis for the selectivity of SpeC-MHC II recognition, and provide one mechanism by how SAgs are capable of distinguishing between different MHC II alleles.

Superantigens (SAGs) interact with host immune receptors to induce a massive release of inflammatory cytokines that can lead to toxic shock syndrome and death. Bacterial SAGs can be classified into five distinct evolutionary groups. Group V SAGs are characterized by the alpha3-beta8 loop, a unique approximately 15 amino acid residue extension that is required for optimal T cell activation. Here, we report the X-ray crystal structures of the group V SAG staphylococcal enterotoxin K (SEK) alone and in complex with the TCR hVbeta5.1 domain. SEK adopts a unique TCR binding orientation relative to other SAG-TCR complexes, which results in the alpha3-beta8 loop contacting the apical loop of framework region 4, thereby extending the known TCR recognition site of SAGs. These interactions are absolutely required for TCR binding and T cell activation by SEK, and dictate the TCR Vbeta domain specificity of SEK and other group V SAGs.