2-O-Sulfated Domains in Syndecan-1 Heparan Sulfate Inhibit Neutrophil Cathelicidin and Promote Staphylococcus aureus Corneal Infection

Abstract

Background: Syndecan-1 promotes bacterial infections, but how this is accomplished remains unclear. Results: Syndecan-1 and 2-O-sulfated heparan compounds specifically enhanced S. aureus corneal virulence and inhibited bacterial killing by CRAMP secreted from degranulated neutrophils. Conclusion: Specific structural motifs in syndecan-1 HS promote S. aureus corneal infection by inhibiting neutrophil CRAMP. Significance: This study uncovers a new pathogenic role for syndecan-1 in bacterial infection.

Isolation and functional analysis of syndecans

“…Several papers describe procedures to isolate neutrophils in detail (Boxio, Bossenmeyer-Pourie, Steinckwich, Dournon, & Nusse, 2004; Hu, 2012; Swamydas, Luo, Dorf, & Lionakis, 2015), and several isolation kits are also commercially available (e.g., Miltenyi Biotec, Cayman Chemical, Stemcell Technologies). Briefly, in the procedure that we use (Hayashida et al, 2015), femurs and tibias are isolated from euthanized mice, cleaned, and flushed with Hank’s Balanced Salt Solution containing 10mM HEPES, pH 7.4. Bone marrow cells are centrifuged at 300× g for 10min, resuspended in 45% Percoll solution, layered on top of a 62% and 81% Percoll gradient solution, and centrifuged at 1500× g for 30min.…”

“…Purified syndecan-1 ectodomains bind and inhibit the capacity of several AMPs to kill bacterial pathogens in an HS-dependent manner (Hayashida et al, 2015; Park et al, 2001). Briefly, the effects of purified syndecans on bacterial killing by AMPs can be determined by incubating bacteria (e.g., Staphylococcus aureus , Pseudomonas aeruginosa from ATCC) with AMPs (e.g., LL-37, CRAMP from Anaspec) without or with increasing doses of purified syndecans, and measuring bacterial survival by plating serial dilutions on nutrient agar plates and counting bacterial colonies (Hayashida et al, 2015; Jinno & Park, 2015).…”

Glycosaminoglycans and infection

“…In contrast, syndecan-1 was found to promote S. aureus corneal infection in an HS-dependent manner (66, 67). S. aureus is one of the leading causes of bacterial keratitis, accounting for 10–25% of confirmed cases (68–70).…”

“…The significance of this mechanism is supported by the findings that ablation of syndecan-1 or inhibition of shedding significantly reduces S. aureus corneal virulence, whereas addition of purified syndecan-1 ectodomains or HS significantly enhances virulence (66), and S. aureus induces syndecan-1 shedding through a-toxin (72), a major virulence factor for S. aureus keratitis (73, 74). Interestingly, inhibition of CRAMP by syndecan-1 HS was found to be dependent on 2- O -sulfate groups (67). Because other major corneal bacterial pathogens, such as Streptococcus pneumoniae and P. aeruginosa , also induce syndecan-1 shedding in epithelial cells (25, 75), subversion of 2- O -sulfated domains in syndecan-1 ectodomain HS to inhibit cationic antimicrobial peptides may be a broadly used pathogenic strategy in bacterial corneal infections.…”

“…Furthermore, mice lacking syndecan-1 develop normally, but show abnormal responses to infectious and inflammatory stimuli. Importantly, these phenotypes can be rescued by HS/HP (26, 67, 118–120), indicating that HS functions are also important post-developmentally. Studies in the last several decades have established that biological functions of GAGs also include modulation of microbial pathogenesis and host defense mechanisms.…”