The classical lancefield antigen of group a Streptococcus is a virulence determinant with implications for vaccine design

Abstract

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.

Figure 1. Schematic Representation of the GAC Gene Cluster, Mutagenesis Scan, and Δ GacI mutant

(A) Schematic representation of the GAS M1T1 strain 5448 group A carbohydrate (GAC) gene cluster M5005_Spy0602-0613, which was renamed gacA-L, and annotated gene functions based on analysis using the SEED tools (URL: pubseed.theseed.org). (B)Latex agglutination reaction with GAC-specific beads and GlcNAc-specific sWGAlectin staining of viable GAS inserional knockout mutants in genes gacD-L. Grey fill: medium control; black line:sWGA-FITC stain. Histogram numbers indicate geometric mean of fluorescence. (C) Latex agglutination reaction with GAC-specific beads and GlcNAc expression as assessed by sWGA stain as indicated on GAS WT, ΔGacImutant and GacI* complemented strain. HPLC tracing and linkage analysis with deduced schematic structure of the repeating unit of extracted GAC from (D) GAS WT and (E)ΔGacI mutant strain. See also Figure S1.

Figure 2. Streptococcus dysgalactiae subsp. equisimilis Testing Positive for GACPossess the gacA-gacK Gene Locus

(A) Circular genome map of the group G carbohydrateexpressing Streptococcus dysgalactiae subsp. equisimilis (SDSE) GGS_124 genome (accession numberAP010935) with BLAST comparisons to the completely sequenced group G carbohydrate expressing SDSE strain SDSE_D166B genome (accession number CP002215) and the draft genomes of two SDSE strains expressing the GAC antigen, 2005-0193 and 2006-0098. The comparative map was created using BRIG. The inner most rings show GC content (black) and GC skew (purple/green) of GGS_124. The three outer rings show BLAST comparisons (using BLASTn and an E-value cutoff of 10.0) to SDSE_D166B (Red) and to the draft genome sequences of 2005-0193 (blue) and 2006-0098 (orange). The legend shows the percent identity of BLASTn hits to the GGS_124 reference. Both genomes share similar genome synteny with both GGS_124 and SDSE_D166B. Labels around the outer ring refer to genomic regions present in GGS_124 but not in the two SDSE strains expressing GAC, including the GAC carbohydrate gene cluster. (B) Genome architecture of the carbohydrate loci located between the dnaG toinfC gene sequences from 2005-0193 with GGS_124 and M1 strain MGAS5005. Regions of genetic similarity were determined using BLAST with graphical representation of syntenic gene content designated using Easyfig 2.1. Purple coding sequences refer to the GAC encoded gacA to gacL cluster. Blue shaded regions between the stacked genome sequences reflect conserved gene content with percenthomology indicated by the legend. (C) Similarity plot showing carbohydrate locus nucleotide sequence conservation between 2005-0193 versus GGS_124 (blue line) and 2005-0193 versus MGAS5005 (green line). Evidence of homologous recombination between a GAS donor and the SDSE progenitor is evident between the gacA and pepT genes. The plot was generated from a clustal alignment of sequence between the dnaG and infC using a sliding window of 100 bp with values plotted as an average over a 100 bp sliding window.

Figure 3. TarO/GbcOHomologue M5005_Spy0240 Contributes to GAC Biosynthesis

Tunicamycin is a specific inhibitor of UDP-GlcNAC:lipid phosphate transferases like TarO and GbcO and produces a similar phenotype to a gbcO knockout in group B Streptococcus (GBS). WT M1 GAS was treated with different concentrations of tunicamycin to inhibit the activity of homologous enzyme encoded by the gacO gene (M5005_Spy0240), which resulted in (A) growth inhibition (mean ± SEM of four independent experiments, one-way ANOVA), (B) increased sensitivity to mutanolysin (100 U/ml; mean ± SEM of two independent experiments, two-way ANOVA), *p< 0.05,**p< 0.01, ***p< 0.001. (C) changes in cell morphology, and (D) complete loss of rhamnose expression indicating a loss of GAC production. Abbreviations: Rha = Rhamnose; Man = Mannose; Glc = Glucose; GlcNAc =N-acetyl-D-glucosamine; PS = polysaccharide; Tun = tunicamycin at indicated concentration (g/ml). See also Figure S2.

Figure 4. GacI Mutant Bacteria Are Not Impaired in Expression of Several Known GAS Virulence Factors or Traits

GAS WT and ΔGacI mutant bacteria were assessed for (A) Growth in Todd-Hewitt broth. (B) M1 protein expression: Grey fill: serum control, black line: WT GAS, black dash: GacI, grey dotted: GacI*reconstituted; (C) Capsule expression in lab grown and animal passaged bacteria (mean ± SEM of two independent experiments); (D) fibrinogen binding; (E)SpeB secretion; (F) plasmin accumulation of GASWT, GacI mutant and GacI*. Pooled normalized data from three independent experiments are shown(mean ± SEM; one-way ANOVA). (G) Carbohydrate metabolism of GAS WT and GacI mutant assessed using diagnostic API 50 CH test (BioMerieux; t = 48 h). Yellow color indicates the specific carbohydrate is fermented, whereas blue/purple color indicates the bacteria do not metabolize the sugar. See also Figure S3.

Figure 5. Antigen-negative GAS Display Increased Sensitivity to Neutrophil and Platelet Immune Defenses

(A, B)ΔGacI mutant bacteria are defective in whole blood proliferation both in the absence (A) and presence (B, +cytochalasin D, CytD 25 μg/ml) of phagocytosis. Data in (B) represent the 2 h time point. Pooled data from three independent experiments are shown (mean ± SEM; one-way ANOVA). (C) Total (no inhibitor) and extracellular (+Cyt D, 10 μg/ml) bacterial killing by isolated human neutrophils. Surviving CFU were quantified after 15 min and 30 min for total killing and extracellular killing, respectively. Combined data from three independent experiments using different donors are shown (Mean ± SEM; one-way ANOVA). (D)Survival of bacteria in neutrophil extracellular traps (NETs). Neutrophils were incubated with 25 nM PMA for 4 h and incubated with bacteria for 30 min. Pooled data from two independent experiments are shown (mean ± SEM, one-way ANOVA). (E) Kinetic analysis showing increased susceptibility to cathelidicin antimicrobial peptide LL-37 for the GacImutant compared to WT. (F) Surface plasmon resonance (SPR) analysis. LL-37 peptide was immobilized on a CM5 sensor chip by amine coupling. Various concentrations of purified WT (left) or GacI mutant GAC (right) were used as analytes to detect binding to LL-37. SPRsensorgrams were generated by subtraction of the reference flow cell and the signals obtained by injection of only the running buffer from the measured response units. (G) Increased hydrophobicity of the GacI mutantbacteria compared to GAS WT as assessed by the n-hexadecane partition assay. The Y-axis indicates the % of original inoculum recovered from the n-hexadecane layer. (H) Survival of GAS WT or ΔGacI mutant strain in 5% complement sufficient or heat-inactivated (HI) baby rabbit serum (BRS). Survival was quantified after 5 h incubation at 37°C. Representative data are shown. (I) Effects of human serum or thrombin-activated platelet supernatant on survival of GAS WT or ΔGacI mutant bacteria. Serum and platelets were collected from the same donor, processed as described in the Material and Methods, and added to a final concentration of 5% and 25%, respectively. Pooled data from 5 independent experiments are shown (mean ± SEM; ratio t-test). *p< 0.05, **p< 0.01, ***p< 0.001. (J) No difference in growth of GAS WT or ΔGacI mutant strain in 5% human plasma (mean ± SEM, t-test). (K) C3b deposition on GAS WT and ΔGacI bacteria after incubation with a range of serum concentrations. Pooled data from 4 independent experiments are shown (mean ± SEM). See also Figure S4.

FIGURE 6. Loss of the GAC GlcNAcSide Chain Attenuates GAS Virulence

(A) Survival curve of rabbits infected with GAS WT or ΔGacI mutant bacteria. Rabbits were infected with 4 × 10⁹ CFU intrabronchially and survival was monitored for seven days (n = 9 rabbits of either sex per group in three independent experiments; log-rank test). (B) Gross lung appearance and microscopic H&E stain of rabbit lungs after infection with GAS WT or ΔGacI mutant bacteria. (C) Body temperature, (D) lung bacterial counts, and (E) lung TNF-α levels in lungs of infected rabbits 12 hours after intrapulmonary challenge; n = 4 rabbits per group; t-test; mean and 95% confidence interval. (F) Survival curve of mice upon systemic infection with GAS WT or ΔGacI mutant bacteria; survival was monitored for 6 days (n = 11 per group; log-rank test). (G) Blood CFU of mice 24 hours post systemic infection (n = 10 mice per group; t-test; mean and 95% confidence interval). *p< 0.05, ***p< 0.001. See also Figure S5.

Figure 7. Antiserum Raised Against GlcNAc-Deficient GAC Promotes Opsonophagocytosis of Multiple GAS Serotypes

(A) GlcNAc-specific human monoclonal antibody (mAb) 3B6 derived from a patient with rheumatic carditis(Galvin et al., 2000) shows significantly reduced cross-reactivity with GlcNAc-deficient GAC. *p< 0.05 (t-test; mean ± SEM). (B) Binding of polyclonal anti-ΔGAC IgG (black line) to WT GAS of 8 different disease-associated serotypes; grey fill = non-immune rabbit IgG control. Serotype is indicated in histogram. (C) Enhanced killing of WT M1 GAS in human whole blood upon addition of anti-ΔGAC rabbit antiserum versus normal rabbit serum (NRS). (D)Improved opsonophagocytic killing of multiple GAS serotypes by isolated human neutrophils upon addition of anti-ΔGAC antiserum versus NRS; mean ± SEM, t-test; *p< 0.05, **p< 0.01, ***p< 0.001, n.s not significant. (E)Mice are protected from infection with WT GAS M49 through passive immunization with ΔGAC antiserum vs. NRS (n = 12 per group; log-rank test). See also Figure S6.