Poster Presentation 50th Lorne Proteins Conference 2025

Insights into the mechanism of the Streptococcus pneumoniae glutathione import system (#142)

Sarah E du Toit 1 , Pierre de Cordovez 1 , Renwick Dobson 2 , Christoph Goebl 1 , Nina Dickerhof 1
  1. University of Otago, Christchurch, New Zealand
  2. School of Biological Sciences, University of Canterbury, Christchurch, New Zealand

Streptococcus pneumoniae has been recognized by the World Health Organization as a significant public health threat, particularly due to its rapid acquisition of antibiotic resistance. Our research demonstrates that S. pneumoniae relies on importing the antioxidant tripeptide glutathione (GSH) from its human host to survive the oxidative stress imposed by the immune system. This GSH import is vital for the pathogen's virulence, highlighting the therapeutic potential of targeting this system. However, a precise understanding of how the import works is lacking.

Our group identified a crucial ATP-binding cassette (ABC) transporter that facilitates GSH import in S. pneumoniae. This transporter contains 1.) a specific GSH substrate binding domain (SBD), GshT, which scavenges GSH from the host, 2.) a transmembrane channel for translocation, and 3.) nucleotide binding domains that drive the transport process.

To investigate the mechanism of GshT-mediated GSH import, we employed a range of biophysical techniques. Differential scanning fluorimetry (DSF) using recombinant GshT and various GSH analogues demonstrated that GSH is the strongest binding partner for GshT and highlighted the critical roles of both carboxyl groups and backbone length of the ligand in determining binding affinity. Further DSF experiments on mutant GshT proteins identified critical residues on the protein involved in GSH binding. Isothermal titration calorimetry (ITC) measurements confirmed that GshT binds both GSH and its oxidized form (GSSG) with affinities in the low µM range. Additionally, solution nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS) revealed conformational changes upon GSH binding, and NMR spectroscopy of mutant GshT proteins showed complete loss of GSH binding.

These findings enhance our understanding of the molecular mechanisms underpinning GSH transport in S. pneumoniae. This research lays the groundwork for the development of novel antimicrobial therapies targeting this major human pathogen.