The molecular basis of GshT substrate binding reveals how Streptococcus pneumoniae acquires the antioxidant glutathione

Glutathione is synthesised across all kingdoms of life, but certain bacteria, including the major pathogen Streptococcus pneumoniae, import host glutathione instead. Glutathione import is known to involve the substrate-binding protein GshT, but the molecular basis underlying glutathione scavenging by pneumococcal GshT remains incompletely understood. We used isothermal titration calorimetry to demonstrate that pneumococcal GshT binds glutathione with low micromolar affinity. Thermal shift assays with glutathione derivatives demonstrated that two adequately spaced carboxylates in glutathione are essential for binding. Monitoring NMR chemical shift perturbations of mutated GshT variants upon glutathione addition revealed that two conserved positively charged residues lining the ligand-binding pocket determine the ability to bind glutathione. Mutagenesis of these residues in S. pneumoniae abolished GSH uptake, impaired growth and lowered oxidant tolerance. Together, our data demonstrate that a lock-and-key charge complementarity drives the GshT-glutathione interaction. Our findings provide insights into pathogen survival mechanisms that may inform strategies to combat infectious disease.