Streptococcus pneumoniae (the pneumococcus) is the leading cause of bacterial pneumonia globally and a World Health Organisation priority pathogen for antimicrobial development. Ionophores, compounds that facilitate unregulated shuttling of metal ions across bacterial membranes leading to toxicity, have garnered interest to potentiate and restore the efficacy of antibiotics against multidrug resistant (MDR) bacterial pathogens, including the pneumococcus. Here, we investigate the development of a combination therapy using the copper ionophore, CuII(GTSM), to break tetracycline (TET) resistance in S. pneumoniae.
The antimicrobial activity of TET and CuII(GTSM) was first determined against a diverse panel of clinically important MDR S. pneumoniae serotypes. Minimal inhibitory concentration (MIC) assays revealed ≥4-fold reduction in the MIC of TET with CuII(GTSM), independent of serotype. To investigate the molecular mechanism(s) underlying this enhanced antimicrobial activity, whole-cell metal accumulation analyses were conducted. These revealed increased copper abundance under CuII(GTSM), which was further potentiated by TET, suggesting a mechanism by which these compounds synergise to enhance cellular copper toxicity.
To determine the impact of enhanced copper accumulation, we applied multi-omic analyses comprising transcriptomics and metabolomics. This showed heavily impacted cellular carbon source utilisation pathways, with broad transcriptomic changes to carbohydrate uptake, coupled with significant changes in metabolite abundance throughout glycolysis and downstream pathways including fatty acid and capsule biosynthesis. To investigate this further, targeted phenotypic characterisations of the pneumococcal cell envelope were conducted. These analyses revealed significantly increased membrane rigidity and reduced capsule production only when TET and CuII(GTSM) were combined. Scanning electron microscopy confirmed maintenance of cellular integrity, suggesting specific Cu-induced perturbation to essential cellular biosynthetic pathways is likely a primary mechanism of TET potentiation.
Collectively, this study provides initial insights into the use of copper ionophores in combination with TET as a therapeutic strategy against MDR S. pneumoniae and provides the foundation for future therapeutic development.