Staphylococcal Superantigen-like protein 11 mediates neutrophil adhesion and motility arrest, a unique bacterial toxin action

www.nature.com/scientificreports OPEN Received: 20 August 2018 Accepted: 5 February 2019 Published: xx xx xxxx Staphylococcal Superantigen-like protein 11 mediates neutrophil adhesion and motility arrest, a unique bacterial toxin action Chen Chen, Chen Yang & Joseph T. Barbieri Methicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen, which causes superficial to lethal clinical infections. Neutrophils are the most abundant leukocytes in the blood and are the first defense mechanism against S. aureus infections. Here we show Staphylococcal Superantigen-Like protein 11 (SSL11) from MRSA USA300_FPR3757 mediated differentiated human neutrophil-like cells (dHL60) motility arrest by inducing cell adhesion and “locking” cells in adhesion stage, without inducing oxidative burst. Pre-incubation of SSL11 with the glycan Sialyl Lewis X blocked SSL11 function and deglycosylation of dHL60 cells by PNGase F abolished SSL11 binding, suggesting that SSL11 functions via interacting with glycans. This is the first description of a bacterial toxin inhibiting neutrophil motility by inducing adhesion and “locking” cells in an adhesion stage. Therefore, this study might provide a new target against S. aureus infections. Staphylococcus aureus (S. aureus) is a major opportunistic pathogen of humans, which causes superficial complications to lethal, invasive infections. Prevalence of methicillin-resistant S. aureus (MRSA) imposes a high burden on healthcare resources. USA300 is the predominant MRSA clone in the US1. S. aureus survival in humans requires evasion of the host immune system, where complement activation and neutrophil-mediated killing are the primary defense mechanisms2. S. aureus Superantigen-Like proteins (SSLs) are not mitogenic to T cells and do not bind MHC class II molecule, despite sharing similar structure with Superantigens (SAgs)3. Not all SSLs functions are known, but SSL activities identified so far involve immune evasion: SSL3, SSL5 and SSL11 inhibit neutrophil activation and rolling4–6; SSL7 and SSL10 bind IgA and IgG and inhibits complement activation7–10. Soluble factor(s) from Lactobacillus reuteri (L. reuteri) RC-14 inhibits S. aureus infections in a rat surgical-implant model by inhibiting S. aureus adhesion to surgical implants11. SSL11 showed a dramatic decrease in expression when S. aureus was co-cultured with L. reuteri RC-14 and recombinant SSL11 reacted with all five convalescent human sera samples from patients with previous S. aureus infections12, suggesting that SSL11 plays an important role for S. aureus infections. Understanding immune modulating protein SSL11 from MRSA might provide new targets against S. aureus infections. Neutrophils are the most abundant leukocytes and the first host immune defense against S. aureus infection. The evasion of host neutrophil recruitment to the site of infection is essential to the success of S. aureus as a pathogen2. Precise regulation of neutrophil adhesion and de-adhesion is essential for migration towards a site of inflammation13. Differentiated HL60 cells (dHL60) are a widely-used model of human neutrophils for migration and chemotaxis14. In the current study, we show for the first time that SSL11 disrupts neutrophil motility by induction of cell adhesion. These findings provide a new therapeutic target against S. aureus infections and neutrophil overstimulated inflammatory diseases. Results SSL11 induces dHL60 cells adhesion and “locks” cells in adhesion stage. In humans, S. aureus survives host immune system by evasion of complement activation and neutrophil-mediated killing2,15. Relative to primary neutrophils, differentiated human HL60 cells (dHL60) are more homogeneous, stable, and more efficient for genetic manipulation. As suspension cells, quiescent dHL60 cells display low adherence. After 30-min Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, 53226, USA. Correspondence and requests for materials should be addressed to C.C. (email: ) Scientific Reports | (2019) 9:4211 | https://doi.org/10.1038/s41598-019-40817-x 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. SSL11 stimulates dHL60 cell adhesion. (A) 2 μg of purified SSL7, SSL11 and SSL113XF were separated by SDS-PAGE and stained with Coomassie Blue. (B) dHL60 cells were incubated with 80 nM of SSL7 or SSL11 in fibronectin (FN)-coated plates at 37 °C for 30 min followed by two PBS washes. Representative DIC images were shown. (C) dHL60 cells were incubated with SSL7 or SSL11 in FN-coated 96-well plates at 37 °C for 30 min followed by two PBS washes. Adherent cells were quantified by crystal violet staining and shown as adhesion arbitrary unit (AU). (D) dHL60 cells were incubated with 80 nM of SSL11 at 37 °C for 30 min and chased in fresh media without SSL11 for another 4 hours in FN-coated plates. Representative DIC images were shown. (E) dHL60 cells were treated with SSL11 as described in (D) in FN-coated 96 well plates. Adherent cells were quantified by crystal violet staining and shown as adhesion arbitrary unit (AU). incubation with SSL11, dHL60 cells transitioned from a non-adhesion to an adhesion phenotype, while untreated cells remained non-adhesion (Fig. 1A,B). A quantitative plate assay showed that SSL11 induced dHL60 cell adhesion in a dose-dependent manner, with 40 nM SSL11 inducing about 50% cell adhesion (Fig. 1C). SSL11 induced adhesion as early as 5 min, with >75% cell adhesion detected by 15 min (Supplementary Fig. S1, Movie 1 and 2). SSL7, which binds IgA and IgG, inhibits complement activation7,9,10, did not mediate dHL60 cell adhesion (Fig. 1B,C), showing the specificity of SSL11-mediated cell adhesion. To test how long cells remained adhesive Scientific Reports | (2019) 9:4211 | https://doi.org/10.1038/s41598-019-40817-x 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 2. SSL11 blocks fMLP-induced dHL60 cell motility. (A) dHL60 cells were incubated with 80 nM of SSL11 at 37 °C for 30 mins in FN-coated plates when fMLP was added and cell motility was recorded for 30 min. Representative motility traces of 20 cells were shown as individual tracks using Ibidi Chemotaxis and Migration Tool. Inserts show hand traced outline of representative images of ctrl cell (small tail) and SSL11-stimulated cell (enlarged adhesive tail). (B) Total length of motility of the 20 cells (μm) shown in (A) was plotted by GraphPad Prism. (C) dHL60 cells were incubated with SSL11 at 37 °C for 30 mins in Fn-coated plates and chased in fresh media without SSL11 for another 4 hrs when fMLP was added and cell motility was recorded for 10 min. Representative motility traces of 20 cells were shown as individual tracks. (D) Total length of motility of the 20 cells (μm) shown in (C) was plotted by GraphPad Prism. after SSL11 treatment, dHL60 cells were incubated with SSL11 for 30 mins, and cells were chased in media without SSL11 fo (...truncated)