Abstract
Streptococcus pneumoniae has been recognised by the World Health Organisation as a major
public health concern, especially considering its rapid rate of acquiring antibiotic resistance.
This bacterium can cause the life-threatening, invasive diseases, pneumonia, meningitis, and
sepsis. S. pneumoniae produces large amounts of hydrogen peroxide during its normal aerobic
metabolism, which is converted to the powerful oxidant hypothiocyanous acid (HOSCN) in the
airway, through the action of host peroxidases. Our group has found that S. pneumoniae has a
high tolerance towards HOSCN, which we recently attributed in part to the glutathione (GSH)
antioxidant tripeptide system. S. pneumoniae lacks GSH biosynthesis genes and instead utilises
an ABC-importer and a GSH substrate-binding protein (GshT) to import the tripeptide from
the environment. S. pneumoniae mutants unable to synthesise GshT have increased
susceptibility to killing by HOSCN and are less virulent in murine infection models. We
therefore propose that GshT is a promising novel drug target.
The aim of this thesis was to gain detailed insights into the interaction between GshT and its
ligands. Binding affinities were measured using isothermal titration calorimetry, and
dissociation constants for GSH and glutathione disulfide (GSSG) binding to GshT were found
to be in the low µM range. Small-angle X-ray scattering provided evidence for a
conformational change upon ligand binding, and nuclear magnetic resonance spectroscopy data
confirmed a structural rearrangement upon binding.
Differential scanning fluorimetry was used to investigate the binding of several GSH
analogues, and GSH was found to be the strongest binder to GshT. The relative binding
capacity of its analogues highlighted the importance for the presence of both N- and C- terminal
carboxyl groups in the ligand, and length of the peptide backbone for binding to GshT.
Differential scanning fluorimetry was also used to investigate the binding capacity of several
mutant GshT proteins to probe which residues in the GshT binding site attribute to binding of
GSH and GSSG.
Collectively, this study provides the molecular basis underlying the function of an antioxidant
importer from a major human pathogen, and we can use the findings from this thesis as a basis
for ligand mimic design.