Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation

Abstract

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20–40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion.

An optimised GAS-pharyngeal cell biofilm model

“…Here we demonstrate an efficacious GAS biofilm-pharyngeal cell model that can support long-term biofilm formation, with biofilms formed resembling those seen in vivo. As per Vyas et al (2020), this model has been used successfully to assess the role of pharyngeal cell surface glycans in mediating GAS biofilm formation 12 . Specifically, the GAS biofilms formed utilising this model have enabled thorough characterisation of GAS biofilm biomass, biofilm viability, EPS matrix content, and EPS associated eDNA and protein content 12 .…”

“…As per Vyas et al (2020), this model has been used successfully to assess the role of pharyngeal cell surface glycans in mediating GAS biofilm formation 12 . Specifically, the GAS biofilms formed utilising this model have enabled thorough characterisation of GAS biofilm biomass, biofilm viability, EPS matrix content, and EPS associated eDNA and protein content 12 . Lastly, GAS biofilms formed by this model have been shown to demonstrate penicillin tolerance profiles of 2500–5000-fold greater than planktonic GAS 12 .…”

“…We also present optimised steps and tips for increased biofilm integrity and reproducibility when performing staining assays like crystal violet. This model has since been used to effectively assess the role pharyngeal cell surface glycans play in GAS biofilm formation and clearly demonstrates the importance of mimicking the epithelial environment in these studies 12 .…”

An Optimised GAS-pharyngeal cell biofilm model

“…Here we demonstrate an efficacious GAS biofilm-pharyngeal cell model that can support long-term biofilm formation, with biofilms formed resembling those seen in vivo . This model has since been used for assessing the role of pharyngeal cell surface glycans in mediating GAS biofilm formation [12]. Hence, the value of this model is that it can be used to explore a plethora of interactions occurring at the GAS-host cell surface interface and the subsequent effects these interactions exert on biofilm formation.…”

“…This model has since been used to effectively assess the role pharyngeal cell surface glycans play in GAS biofilm formation and clearly demonstrates the importance of mimicking the epithelial environment in these studies [12]. [15] *Inoculums listed as ratios (bacteria: bacterial media), growth phase, or optical density.…”