The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function:SElX Inhibits Neutrophil Function

Bacterial superantigens (SAgs) cause Vβ-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vβ-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis

The Staphylococcus aureus iron-regulated surface determinant A (IsdA) increases SARS CoV-2 replication by modulating JAK-STAT signaling WESTERN BLOTS

western blot images related to Figure S3, S6 and S8

The Staphylococcus aureus iron-regulated surface determinant A (IsdA) increases SARS CoV-2 replication by modulating JAK-STAT signaling WESTERN BLOTS

western blot images related to Figure S3, S6 and S8.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

Immunological homeostasis at the ovine placenta may reflect the degree of maternal foetal interaction

Successful mammalian pregnancies are a result of complex physiological, endocrinological and immunological processes that combine to create an environment where the mother is tolerant to the semi-allogeneic fetus. Our knowledge of the mechanisms thatcontribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as thisis the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorialplacentation found in other species such as ruminants. Here, we examine three features associated with reproductive immuneregulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: majorhistocompatibility complex (MHC) expression, Indoleamine 2, 3 dioxygenase-1 (IDO-1) expression and Natural Killer (NK) cellinfiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHCclass I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHCalleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence ofconstitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from thematerno-fetal interface were negative for cells expressing NKp46/NCR1. Collectively, these observations suggest that the relatively non-invasive synepitheliochorial placentation found in sheep has a more limited requirement for local immunoregulation compared to the more invasive haemochorial placentation of primates and rodents

The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function

Bacterial superantigens (SAgs) cause Vβ-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vβ-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis

Neutrophil binding-deficient mutants of SElX retain mitogenic activity.

<p>Isolated human PBMC were stimulated with recombinant SElX and sialic acid-binding mutants. After 72 h incubation, proliferation was determined by analysing the incorporation of [³H] thymidine. Results shown are the means of triplicate measurement from 3 human donors ± standard deviation of the mean.</p

SElX does not contribute to <i>S. aureus</i> virulence in a mouse skin abscess infection model.

<p>(i) Lesion size of each mouse (n = 10) was measured every 24 h post inoculation (p.i.) for 6 d. Mean lesion size ± SEM is plotted for each of the 4 USA300 LAC mutant groups. (ii) Bacteria were recovered from excised skin lesions and enumerated by serial dilutions. CFU were normalised to the weight of tissue homogenised to give the bacterial load per mg of tissue. CFU per mg are displayed for each infected animal; the horizontal line indicates the mean CFU/mg of tissue and vertical bars show the SEM for each group (n = 10) (iii) Representative images from histological examinations of skin lesions 72 h and 144 h post inoculation. Mounted sections were stained with haematoxylin and eosin. Black arrows on each image indicate the surface of the epidermis.</p

SElX binds to human monocytes and neutrophils from multiple mammalian species.

<p>Flow cytometry analysis of recombinant staphylococcal proteins binding to isolated human cells. Cell type was determined by forward (FSC-H) and sideways (SSC-H) scatter (i). SElX binding to human neutrophils (ii) monocytes (iii) and lymphocytes (iv) in addition to neutrophils isolated from mice (v), cattle (vi) and rabbits (vii), was examined. For all graphs binding was detected using mouse anti-HIS-FITC IgG binding to the 6 x HIS-tag on the recombinant proteins. Mean median fluorescence of three donors is shown ± standard error of the mean (SEM). SSl5 and SSl7 were used as positive and negative controls, respectively. The same legend is used for all graphs.</p

SElX protein sequence encodes a conserved sialic acid-binding motif.

<p>(i) A conserved glycan binding motif (colored red) is present in the amino acid sequences of characterised SSl-proteins and SElX. (ii) The sialic acid-binding motif is conserved in all 17 alleles of SElX. (iii) Amino acid conservation across the sialic acid-binding region of 7 staphylococcal neutrophil binding proteins (SSl2, SSl3, SSl4 SSl5, SSl6, SSl11 and SElX). The probability of residues at each position is proportional to the size of the letters. Image generated using the weblogo 3.4 program (<a href="http://weblogo.threeplusone.com/" target="_blank">http://weblogo.threeplusone.com/</a>). Chemical property color scheme: green is polar, blue is basic, red is acidic, purple is neutral and black is hydrophobic. Sequences were obtained from previously published work [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006461#ppat.1006461.ref006" target="_blank">6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006461#ppat.1006461.ref023" target="_blank">23</a>].</p