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.

Keywords: EPS; Streptococcus pyogenes; antibiotics; biofilm; glycans; group A Streptococcus; penicillin.

Figure 1

Indiscriminate removal of N-linked glycans from the pharyngeal cell surface via PNGase F treatment results in increased M12 Group A Streptococcus (GAS) biofilm biomass. (A) Assay schematic for 72 h M12 GAS biofilms formed on PNGase F pre-treated and untreated pharyngeal monolayers. (B) Initial adherence enumerated for planktonic GAS following 2 h incubation with Detroit-562 cell monolayers. 72 h biofilms were assessed for (C) biofilm biomass via crystal violet staining and (D) colony forming units via enumeration. Data represents mean ± SEM, with statistical analysis performed, * (p ≤ 0.05) and **** (p ≤ 0.0001); n = 3 biological replicates, with 3 technical replicates each.

Figure 1

Indiscriminate removal of N-linked glycans from the pharyngeal cell surface via PNGase F treatment results in increased M12 Group A Streptococcus (GAS) biofilm biomass. (A) Assay schematic for 72 h M12 GAS biofilms formed on PNGase F pre-treated and untreated pharyngeal monolayers. (B) Initial adherence enumerated for planktonic GAS following 2 h incubation with Detroit-562 cell monolayers. 72 h biofilms were assessed for (C) biofilm biomass via crystal violet staining and (D) colony forming units via enumeration. Data represents mean ± SEM, with statistical analysis performed, * (p ≤ 0.05) and **** (p ≤ 0.0001); n = 3 biological replicates, with 3 technical replicates each.

Figure 2

Visual inspection of 72 h M12 GAS biofilms captured via SEM revealed substantial extracellular polymeric substances (EPS) present in biofilms formed on PNGase F pre-treated pharyngeal cell monolayers. Images are representative of biofilms formed on (A) untreated and (C) PNGase F pre-treated pharyngeal monolayers. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS matrix material present (big and small black arrows). SEM images of (B) untreated and (D) PNGase F pre-treated Detroit 562 pharyngeal cell monolayers (without biofilm) are also included. Biofilms and Detroit 562 pharyngeal cell monolayers (without biofilm) were imaged using the JEOL JSM-7500 microscope at 15,000× and 500× magnification, respectively. SEM images were randomly selected and represent two biological replicates with two technical replicates each.

Figure 2

Visual inspection of 72 h M12 GAS biofilms captured via SEM revealed substantial extracellular polymeric substances (EPS) present in biofilms formed on PNGase F pre-treated pharyngeal cell monolayers. Images are representative of biofilms formed on (A) untreated and (C) PNGase F pre-treated pharyngeal monolayers. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS matrix material present (big and small black arrows). SEM images of (B) untreated and (D) PNGase F pre-treated Detroit 562 pharyngeal cell monolayers (without biofilm) are also included. Biofilms and Detroit 562 pharyngeal cell monolayers (without biofilm) were imaged using the JEOL JSM-7500 microscope at 15,000× and 500× magnification, respectively. SEM images were randomly selected and represent two biological replicates with two technical replicates each.

Figure 3

Structures bearing terminal mannose predominate the surface of Detroit 562 pharyngeal cells as determined by PGC-LC-ESI-MS/MS. (A) Surface N-glycans are primarily oligomannose structures. (B) Mannose is the most abundant terminal monosaccharide of N-glycans. (C) Examples of oligomannose, complex, and hybrid N-glycans are provided, including relative abundance and mass-to-charge ratio (m/z) as detected by MS. Structures were identified primarily using MS² spectra (D–F) [28,29,30,31] in addition to precursor mass:charge ratio (m/z) and retention time. Structural isomers sharing the same m/z, composition, and terminal monosaccharide presentation were combined in evaluation of abundance, calculated by integration of area under of the curve from extracted ion chromatograms. Abundance values are relative and are presented as combined mean ± SEM from 3 biological replicates, each with 3 technical replicates. * Denotes doubly charged fragments. Glycans are represented using conventional graphical nomenclature [32].

Figure 4

Pre-treatment of pharyngeal cell surface with α1-6 mannosidase and Sialidase A results in significantly increased M12 GAS biofilm biomass. (A) Assay schematic for the characterization of biofilms formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase (A) pre-treated pharyngeal monolayers vs. untreated. (B) Initial adherence enumerated for planktonic GAS upon 2 h incubation. 72 h biofilms are assessed for (C) biofilm biomass via crystal violet staining and (D) colony forming units via enumeration. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * (p ≤ 0.05); n = 3 biological replicates, with 3 technical replicates each.

Figure 4

Pre-treatment of pharyngeal cell surface with α1-6 mannosidase and Sialidase A results in significantly increased M12 GAS biofilm biomass. (A) Assay schematic for the characterization of biofilms formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase (A) pre-treated pharyngeal monolayers vs. untreated. (B) Initial adherence enumerated for planktonic GAS upon 2 h incubation. 72 h biofilms are assessed for (C) biofilm biomass via crystal violet staining and (D) colony forming units via enumeration. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * (p ≤ 0.05); n = 3 biological replicates, with 3 technical replicates each.

Figure 5

Biofilm EPS increases significantly for biofilms formed on α1-6 mannosidase and Sialidase A pre-treated pharyngeal cell surfaces. (A) Assay schematic for the assessment of biofilm EPS resulting from biofilm formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase A) pre-treated pharyngeal monolayers vs. untreated. 72 h biofilms were assessed for (B) EPS via DMMB staining of sulphated GAGs and (C) EPS associated components (eDNA and protein) via fluorescent staining with Sytox Blue and FilmTracer SYPRO Ruby biofilm matrix stain, respectively. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * (p ≤ 0.05) and ** (p ≤ 0.01); n = 3 biological replicates, with 3 technical replicates each.

Figure 6

Visual inspection of 72 h M12 GAS biofilms captured via SEM revealed substantial EPS present in biofilms formed on exoglycosidase pre-treated pharyngeal cell monolayers. Images are representative of biofilms formed on (A) untreated, (C) α1-6 mannosidase, (E) α1-2,3 mannosidase, and (G) Sialidase A pre-treated pharyngeal monolayers. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS matrix material present (big and small black arrows). SEM images of (B) untreated, (D) α1-6 mannosidase, (F) α1-2,3 mannosidase, and (H) Sialidase A pre-treated Detroit 562 pharyngeal cell monolayers (without biofilm) are also included. Biofilms and Detroit 562 pharyngeal cell monolayers (without biofilm) were imaged using the JEOL JSM-7500 microscope at 15,000× and 500× magnification, respectively. SEM images were randomly selected and represent two biological replicates with two technical replicates each.

Figure 6

Visual inspection of 72 h M12 GAS biofilms captured via SEM revealed substantial EPS present in biofilms formed on exoglycosidase pre-treated pharyngeal cell monolayers. Images are representative of biofilms formed on (A) untreated, (C) α1-6 mannosidase, (E) α1-2,3 mannosidase, and (G) Sialidase A pre-treated pharyngeal monolayers. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS matrix material present (big and small black arrows). SEM images of (B) untreated, (D) α1-6 mannosidase, (F) α1-2,3 mannosidase, and (H) Sialidase A pre-treated Detroit 562 pharyngeal cell monolayers (without biofilm) are also included. Biofilms and Detroit 562 pharyngeal cell monolayers (without biofilm) were imaged using the JEOL JSM-7500 microscope at 15,000× and 500× magnification, respectively. SEM images were randomly selected and represent two biological replicates with two technical replicates each.

Figure 7

Assessment of the effect exoglycosidase treated pharyngeal cell monolayers exert on biofilms of eight other GAS M-types. Biofilm biomass was quantified via crystal violet staining. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test, * (p ≤ 0.05), ** (p ≤ 0.01), and *** (p ≤ 0.001); n = 3 biological replicates, with 3 technical replicates each.