Structure of the Streptococcus pyogenes NAD ⁺ Glycohydrolase Translocation Domain and Its Essential Role in Toxin Binding to Oropharyngeal Keratinocytes

Abstract

The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus , GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of co-toxins streptolysin O (SLO) and NAD + -glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a β-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro , suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. Importance NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus , the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.

Pathogenesis, epidemiology and control of Group A Streptococcus infection

“…SLO and NADase physically interact and co-stabilize after secretion 92 . NADase-dependent membrane binding promotes pore formation by SLO 93 , which conversely facilitates translocation of NADase into host cells 94 . In combination, SLO and its co-toxin NADase promote GAS intracellular survival and cytotoxicity in macrophages and epithelial cells 95,96 , impair host defences in these cell types through Golgi fragmentation 97 and contribute to pathogenesis in vivo 98 .…”

Structural basis underlying the synergism of NADase and SLO during group A Streptococcus infection

“…To further understand the structural details of how the N-terminal translocation domain of NADase, which is missing in our determined crystal structure, interacts with SLO, we performed SAXS-based atomistic modeling of the full-length NADase and NADase/SLO complex. Currently, only partial atomic structures of NADase are available: the C-terminal domain in complex with IFS 35 , 36 and a recently determined structure of the N-terminal domain by nuclear magnetic resonance 40 . To delineate how the domains are linked, a structural model of full-length NADase was generated by AlphaFold 41 .…”

“…8a ), supporting the notion that the preservation of the NADase/SLO complex plays an important role in the molecular evolution of NADase 46 . Moreover, a recent report shows NADase residues, Tyr120, Tyr121, Tyr151, and His177, are involved in mediating carbohydrate recognition and membrane binding 40 . These residues are located at the solvent-exposed area in the NADase/SLO multistate model (Supplementary Fig.…”

Immunomodulating Enzymes from Streptococcus pyogenes—In Pathogenesis, as Biotechnological Tools, and as Biological Drugs

“…The crystallized structure only contains the C-terminal catalytic domain. ( B ) The structure for the N-terminal domain as determined by nuclear magnetic resonance (NMR) spectroscopy, PDB ID: 7JI1 [ 82 ] shown in rainbow colors.…”