Streptococcus pneumoniae (the pneumococcus) is a significant global pathogen, responsible for more than one million deaths each year, primarily in the developing world. 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, THP-1 macrophage survival 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 less viable in multiple niches compared to the single ΔcopA and ΔgshT mutants. Interestingly, even in macrophages, where it is postulated that invading pathogens are directly exposed to high concentrations of copper, survival of D39ΔcopA was equivalent to wildtype, with decreased survival observed only for D39ΔgshT and D39ΔcopAΔgshT.
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.