Abstract：
Corynebacterium diphtheriae is the causative agent of diphtheria, a severe respiratory disease in humans. C. diphtheriae colonizes the human upper respiratory tract, where it acquires zinc, an essential metal required for survival in the host. While the mechanisms for zinc transport by C. diphtheriae are not well characterized, four putative zinc ABC-type transporter loci were recently identified in strain 1737: iutABCD/E (iut), znuACB (znu), nikABCD1 (nik1), and nikABCD2 (nik2). A mutant deleted for all four loci (Δ4) exhibited similar growth to that of the wild-type strain in a zinc-limited medium, suggesting there are additional zinc transporters. Two additional gene loci predicted to be associated with metal import, mntABCD (mnt) and sidAB (sid), were deleted in the Δ4 mutant to construct a new mutant designated Δ6. The C. diphtheriae Δ6 mutant exhibited significantly reduced growth under zinc limitation relative to the wild type, suggesting a deficiency in zinc acquisition. Strains retaining the iut, znu, mnt, or sid loci grew to near-wild-type levels in the absence of the other five loci, indicating that each of these transporters may be involved in zinc uptake. Plasmid complementation with cloned iut, znu, mnt, or nik1 loci also enhanced the growth of the Δ6 mutant. Quantification of intracellular zinc content by inductively coupled plasma mass spectrometry was consistent with reduced zinc uptake by Δ6 relative to the wild type and further supports a zinc uptake function for the transporters encoded by iut, znu, and mnt. This study demonstrates that C. diphtheriae zinc transport is complex and involves multiple zinc uptake systems.IMPORTANCEZinc is a critical nutrient for all forms of life, including human bacterial pathogens. Thus, the tools that bacteria use to acquire zinc from host sources are crucial for pathogenesis. While potential candidates for zinc importers have been identified in Corynebacterium diphtheriae from gene expression studies, to date, no study has clearly demonstrated this function for any of the putative transporters. We show that C. diphtheriae encodes at least six loci associated with zinc import, underscoring the extent of redundancy for zinc acquisition. Furthermore, we provide evidence that a previously studied manganese-regulated importer can also function in zinc import. This study builds upon our knowledge of bacterial zinc transport mechanisms and identifies potential targets for future diphtheria vaccine candidates.
摘要：
白喉棒杆菌是白喉的病原体，人类严重的呼吸道疾病。C.白喉定植于人类上呼吸道，在那里它获得锌，在宿主中生存所需的必需金属。虽然白喉梭菌的锌转运机制尚未得到很好的表征，最近在1737菌株中鉴定出四个推定的锌ABC型转运蛋白基因座：iutABCD/E（iut），znuACB(znu),nikABCD1(nik1),和nikABCD2（nik2）。所有四个基因座（Δ4）缺失的突变体在锌限制的培养基中表现出与野生型菌株相似的生长，表明有更多的锌转运蛋白。预测另外两个基因位点与金属进口有关，mntABCD(mnt)和sidAB(sid)，在Δ4突变体中缺失以构建称为Δ6的新突变体。白喉梭菌Δ6突变体在锌限制下相对于野生型表现出显著降低的生长,表明锌的获取不足。保持iut的应变，znu,mnt,或者在没有其他五个基因座的情况下，sid基因座增长到接近野生型水平，表明这些转运蛋白中的每一个都可能参与锌的摄取。用克隆的iut互补质粒，znu,mnt,或nik1基因座也增强了Δ6突变体的生长。通过电感耦合等离子体质谱法定量细胞内锌含量与相对于野生型的Δ6减少的锌摄取一致，并进一步支持iut编码的转运蛋白的锌摄取功能，znu,还有mnt.这项研究表明，白喉梭菌锌转运是复杂的，涉及多个锌吸收系统。IMPORTANCE锌是所有生命形式的关键营养素，包括人类细菌病原体。因此，细菌用于从宿主来源获取锌的工具对于发病机制至关重要。虽然从基因表达研究中已经在白喉棒杆菌中确定了锌导入体的潜在候选者，到目前为止,没有研究清楚地证明任何推定的转运蛋白的这种功能。我们显示白喉梭菌编码至少六个与锌输入相关的基因座，强调锌采集的冗余程度。此外，我们提供的证据表明，先前研究的锰管制进口商也可以在锌进口中发挥作用。这项研究基于我们对细菌锌转运机制的了解，并确定了未来白喉候选疫苗的潜在靶标。
