Binding of NAD+-Glycohydrolase to Streptolysin O Stabilizes Both Toxins and Promotes Virulence of Group A Streptococcus

Abstract

The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD⁺-glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations in nga, the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence.IMPORTANCE The global increase in invasive GAS infections in the 1980s was associated with the emergence of an M1T1 clone that harbors a 36-kb pathogenicity island, which codes for increased expression of toxins SLO and NADase. Polymorphisms in NADase that render it catalytically inactive can be detected in clinical isolates, including invasive strains. However, such isolates continue to produce full-length NADase. The rationale for this observation is not completely understood. This study characterizes the binding interaction between NADase and SLO and reports that the expression of each toxin is crucial for maximal expression and stability of the other. By this mechanism, the presence of both toxins increases toxicity to keratinocytes and is predicted to enhance GAS survival in the human host. These observations provide an explanation for conservation of full-length NADase expression even when it lacks enzymatic activity and suggest a critical role for binding of NADase to SLO in GAS pathogenesis.

Keywords: NAD+-glycohydrolase; Streptococcus pyogenes; cholesterol-dependent cytolysin; pore-forming toxins; streptolysin O.

FIG 1

Inactive NADase can support SLO-mediated cell toxicity. (A) Cell membrane damage to human oropharyngeal keratinocytes due to SLO and NADase was assessed by uptake of a fixable viability dye (blue) after 1.5-h exposure to live GAS (green). Keratinocyte nuclei are stained with propidium iodide (red). The percentage of permeabilized cells is indicated for wild-type GAS 854 and isogenic mutant strains (Table 1 shows strain phenotypes). (B) Quantification of microscopic data is represented from three independent experiments. Data represent the mean ± standard deviation. The significance of differences between groups was evaluated by one-way analysis of variance with Tukey’s posttest (*, P < 0.05; NS, not significant).

FIG 2

NADase supports SLO protein abundance. (A and B) The abundance of SLO was evaluated by Western blotting (A) and hemolytic activity (B) of culture supernatants of Δnga, nga(stop), and nga(G330D) GAS strains; Table 1 shows strain phenotypes. (C) Transcript abundance of slo for GAS strain 854 derivatives. Data are normalized to the wild-type (wt) strain and represent the mean ± standard deviation from three experiments. The significance of differences between groups was evaluated by one-way analysis of variance with Tukey’s posttest (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 3

SLO prevents proteolytic cleavage of NADase. (A) Western blot using antiserum to NADase revealed two distinct bands in culture supernatant of a mutant strain deficient in SLO production (Δslo) but not in supernatants from the wild type or a mutant producing NADase G330D. (B) NADase enzymatic activity associated with the Δslo mutant was not significantly different from that of wild-type 854 as determined by one-way analysis of variance with Tukey’s posttest. (C) NADase cleavage in the Δslo mutant is zinc dependent; it is inhibited by EDTA and enhanced by the addition of ZnCl₂ (1 mM) but not MgCl₂ (1 mM) or MnCl₂ (1 mM). Experiments were performed three times, and where appropriate, the mean ± standard deviation is represented (U, untreated; PI, protease inhibitor). (D) The NADase cleavage site in the Δslo mutant was determined by extraction of full-length and processed NADase from SDS-PAGE and analysis by tryptic digest and LC–MS-MS for fragment identification. The processing site was found to be between amino acids T53 and K54 (vertical arrow). The region of the protein identified in the full-length NADase but not the processed form is highlighted in red. Underlining denotes peptides identified in the analysis for both forms of NADase.

FIG 4

Analytical gel filtration reveals binding of NADase to SLO. (A) NADase and SLO were combined at final concentrations of 30 μM and were loaded on a Superdex 200 Increase 10/300 GL column. Both SLO and NADase could be recovered, as seen by Coomassie blue staining, from a peak that shifted to an earlier elution volume compared to that for either protein alone, suggesting an increase in size from NADase binding to SLO. (B) The shift in elution volume was concentration dependent, as demonstrated by keeping a constant concentration of NADase (25 μM) and incrementally increasing the SLO concentration. (C) A similar shift in elution volume was observed with SLO and the catalytically inactive NADase G330D. (D) Pneumolysin (Ply), a cytolysin from S. pneumoniae, failed to bind NADase at a 30 μM concentration, and thus, no shift for the NADase peak was observed. Representative chromatograms of at least three independent experiments are shown.

FIG 5

SLO and NADase bind in a 1:1 stoichiometry. (A) Molecular masses of NADase (47.2 kDa), SLO (64.6 kDa), and NADase + SLO (83 kDa) were determined by SEC-MALS of column elution peaks. Elution profiles are shown (solid lines) with calculated masses (circles) for each condition. (B and C) The stoichiometry of NADase-SLO binding was determined to be 1:1 by cross-linking of interacting NADase and SLO and identification of a heterodimer species by SDS-PAGE (*) (B) and MALDI-TOF mass spectrometry (C).

FIG 6

Biolayer interferometry (BLI) analysis of NADase binding to SLO. (A) Kinetic analyses of the interaction between full-length NADase and SLO were performed using biolayer interferometry. SLO was immobilized on a biosensor tip, and the binding of incremental 2-fold increases in concentrations of NADase from 312.5 nM to 20 μM was observed. The association and dissociation phases of the experiment are shown. Individual response curves are shown for increasing NADase concentrations. The data were fitted to a biphasic binding model (overlaid in red). (B) An overall dissociation constant (K_D) of 2.58 μM ± 0.38 μM was determined from steady-state analysis of the BLI response versus NADase concentration determined late in the association phase. All experiments were repeated three times. Error bars represent standard deviations.

FIG 7

The C-terminal catalytic domain (190NADase) but not the N-terminal translocation domain (NT194NADase) binds independently to SLO as determined by analytical gel filtration. (A) Analytical gel filtration on a Superdex 200 Increase 10/300 GL column revealed a shift in the elution volume for the catalytic domain of NADase (190NADase) in the presence of SLO. SDS-PAGE and Coomassie blue staining of the peak fraction showed bands corresponding to both SLO and 190NADase. (B) A similar shift in elution volume was not seen for the translocation domain (NT194NADase) in the presence of SLO.

FIG 8

Kinetic analysis of 190NADase binding to SLO. SLO was immobilized on a biosensor tip, and the binding of 190NADase was assessed by BLI at incremental 2-fold increases in concentration. (A) Individual response curves are shown for 190NADase concentrations from 1.25 μM to 80 μM. The association and dissociation phases of the experiment are shown. The data fit a 1:1 binding model (red overlay) between 190NADase and SLO with a pattern suggestive of some nonspecific binding at the highest concentrations of 190NADase. (B) At concentrations of 1.25 to 10 μM 190NADase, a 1:1 binding model (red overlay) was in agreement with the data. (C) Steady-state analysis of the BLI response versus concentration of 190NADase yielded a K_D of 20.65 ± 1.70 μM. (D) A comparison of saturation curves for NADase and 190NADase demonstrates higher affinity between SLO and full-length NADase than between SLO and the 190NADase catalytic domain. All experiments were repeated three times. Error bars represent standard deviations.