Streptococcus pneumoniae (the pneumococcus) is a significant global pathogen, responsible for more than one million deaths each year. As a strictly host-adapted pathogen, the pneumococcus is beholden to the host environment which constitutes its extracellular milieu. As such, essential homeostatic pathways, such as those involved with metal ion homeostasis, have been evolutionarily tuned by the metal stresses encountered at the host-pathogen interface. This includes a high affinity copper efflux pathway, CopA, thought to resist copper intoxication during infection.
Here, we show that during systemic infection, murine tissue copper concentrations increase in numerous niches. However, an isogenic D39ΔcopA strain does not show significant differences in bacterial load in these niches compared to the wildtype. Interestingly, our in vitro analyses show that S. pneumoniae does not modulate copper uptake, but rather accumulates copper relative to the environmental abundance. This suggests that, during infection, increased intracellular copper is being buffered within the cytoplasm, possibly on glutathione, to prevent copper toxicity. We therefore investigated a D39ΔcopAΔgshT strain (lacking copper efflux and glutathione) in in vitro growth assays and in a murine infection model. Growth assays show that D39ΔcopAΔgshT was hyper-susceptible to copper stress, indicating a central role for glutathione in copper buffering. This increased sensitivity to copper was also observed in the infection model, with D39ΔcopAΔgshT significantly perturbed in multiple niches compared to the single ΔcopA and ΔgshT mutants. Notably, by applying novel bioimaging techniques to the infected murine lung, we have now mapped copper distribution throughout the tissue, identifying copper ‘hot spots’, which contain 150-fold more copper than naïve lungs.
Collectively, these data show that host-imposed copper stress is well-tolerated by the pneumococcus providing glutathione is available. These findings also have significant implications for copper-based antimicrobial measures, which are likely to have limited efficacy against S. pneumoniae while glutathione can be actively acquired.