Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas Health Science Center, Houston, Texas, United States of America.
MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, Texas, United States of America.
PLoS Pathog. 2024 Oct 3;20(10):e1012165. doi: 10.1371/journal.ppat.1012165. eCollection 2024 Oct.
Zinc is a vital transition metal for all bacteria; however, elevated intracellular free Zn levels can result in mis-metalation of Mn-dependent enzymes. For Mn-centric bacteria such as Streptococcus pneumoniae that primarily use Mn instead of Fe as an enzyme cofactor, Zn is particularly toxic at high concentrations. 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 bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the ΔccnABCDE 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 ΔccnABCDE strain was not altered by deletion of sodA, whereas the ccn+ ΔsodA strain phenocopied the ΔccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae.
锌是所有细菌的重要过渡金属；然而，细胞内游离锌水平的升高会导致锰依赖酶的错误金属化。对于以锰而非铁作为酶辅因子的锰中心细菌（如肺炎链球菌），高浓度的锌尤其有毒。在这里，我们报告了我们对 CiaRH 调节的五个同源 Ccn sRNAs 在控制肺炎链球菌毒力和金属内稳态中的功能的鉴定和表征。我们表明，从肺炎链球菌菌株 D39（血清型 2）和 TIGR4（血清型 4）中删除所有五个 ccn 基因（ccnA、ccnB、ccnC、ccnD 和 ccnE）会导致锌超敏性，并在小鼠侵袭性肺炎模型中降低毒力。我们提供的证据表明，缺乏五个 ccn 基因的肺炎链球菌菌株中生物可利用的锌不成比例地增加。与对锌中毒或相对高的细胞内游离锌水平的反应一致，编码 CzcD 锌外排泵和锰独立核糖核苷酸还原酶 NrdD-NrdG 的基因的表达在ΔccnABCDE 突变体中相对于其同源 ccn+亲本菌株增加。由于 ccn 基因缺失而导致的锌抑制生长可以通过补充锰或 Oxyrase（一种去除溶解氧的试剂）来挽救。最后，我们发现，缺失 sodA 不会改变ΔccnABCDE 菌株对 Zn 的生长抑制作用，而 ccn+ΔsodA 菌株则表现出与ΔccnABCDE 菌株相同的表型。总的来说，我们的结果表明，Ccn sRNAs 在防止肺炎链球菌锌中毒方面起着至关重要的作用。
