The five homologous CiaR-controlled Ccn sRNAs ofStreptococcus pneumoniaemodulate Zn-resistance

Abstract

ABSTRACTZinc is a vital transition metal forStreptococcus pneumoniae, but is deadly at high concentrations. Zn intoxication ofS. pneumoniaeresults 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 controllingS. pneumoniaevirulence and metal homeostasis. We show that deletion of all fiveccngenes (ccnA,ccnB,ccnC,ccnD, andccnE) fromS. pneumoniaestrains 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 ΔccnABCDEmutants. 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 ΔccnABCDEmutant relative to its isogenicccn+parent strain. The growth inhibition by Zn that occurs as the result of loss of theccngenes 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 ΔccnABCDEstrain was not altered by deletion ofsodA, whereas theccn+ΔsodAstrain phenocopied the ΔccnABCDEstrain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing oxidative stress inS. pneumoniaeduring exposure to excess Zn by modulating Mn uptake.AUTHOR SUMMARYZn and Mn are essential micronutrients for many bacteria, includingStreptococcus pneumoniae. While Zn performs vital structural or catalytic roles in certain proteins, in excess, Zn can inhibit Mn uptake byS. pneumoniaeand displace, but not functionally replace Mn from key enzymes including superoxide dismutase A (SodA). Here, we show that the Ccn small regulatory RNAs promoteS. pneumoniaeMn uptake and resistance to the oxidative stress. Furthermore, we demonstrate that these small regulatory RNAs modulate the ability ofS. pneumoniaeto cause invasive pneumonia. Altogether, these findings reveal a new layer of regulation ofS. pneumoniaeZn and Mn homeostasis and suggest that there are factors in addition to known transporters that modulate intracellular Mn levels.