The five homologous CiaR-controlled Ccn sRNAs of Streptococcus pneumoniae modulate Zn-resistance

Zinc is a vital transition metal for Streptococcus pneumoniae, but is deadly at high concentrations. Zn intoxication of S. pneumoniae results from a deficiency in Mn, which is required for key metabolic enzymes and defense against oxidative stress. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that addition of Zn disproportionately impairs Mn uptake by the ∆ccnABCDE mutants. Consistent with a response to Mn starvation, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the ∆ccnABCDE mutant relative to its isogenic ccn(+) parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or Oxyrase(™), a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the ∆ccnABCDE strain was not altered by deletion of sodA, whereas the ccn(+) ∆sodA strain phenocopied the ∆ccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing oxidative stress in S. pneumoniae during exposure to excess Zn by modulating Mn uptake.