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.
Highlights
Group A Streptococcus (Streptococcus pyogenes; GAS) is a Gram-positive human pathogen responsible for a variety of infections
extracellular polymeric substances (EPS) associated sulphated GAGs increased significantly for M12 GAS biofilms formed on α1-6 mannosidase (p ≤ 0.01) and Sialidase A (p ≤ 0.05) pre-treated pharyngeal cell monolayers compared to the untreated control (Figure 5B)
Penicillin is the antibiotic of choice due to its narrow safety, and accessibility [35]
Summary
Group A Streptococcus (Streptococcus pyogenes; GAS) is a Gram-positive human pathogen responsible for a variety of infections. In a study assessing GAS isolated from pharyngitis patients non-responsive to antibiotics, all 99 GAS isolates demonstrated biofilm-forming ability in vivo, with 60% of these displaying increased penicillin tolerance once in the biofilm phenotype [3]. Most of these studies utilized abiotic surfaces (glass, plastic, polystyrene) and very few incorporated surface coating with extracellular matrix components such as fibronectin, fibrinogen, laminin, or collagen [8–11]. Investigation of glyco-interactions at the GAS-tissue interface in the context of biofilms will enhance our understanding of GAS biofilms and GAS pathogenesis Findings of such studies may inform and support the development of novel anti-biofilm strategies as well as biofilm-specific antibiotic treatments. We examine the role of human pharyngeal N-glycans in M12 GAS biofilm formation and subsequent penicillin tolerance
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