Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
J Bacteriol. 2021 Oct 12;203(21):e0019621. doi: 10.1128/JB.00196-21. Epub 2021 Aug 9.
The acquisition of hemin iron from hemoglobin-haptoglobin (Hb-Hp) by Corynebacterium diphtheriae requires the iron-regulated surface proteins HtaA, ChtA, and ChtC and the recently identified Hb-Hp-binding protein, HbpA. We previously showed that a purified form of HbpA (HbpA-S), lacking the C-terminal region, was able to bind Hb-Hp. In this study, we show that the C-terminal region of HbpA significantly enhances binding to Hb-Hp. A purified form of HbpA that includes the C-terminal domain (HbpA-FL) exhibits much stronger binding to Hb-Hp than HbpA-S. Size exclusion chromatography (SEC) showed that HbpA-FL as well as HtaA-FL, ChtA-FL, and ChtC-FL exist as high-molecular-weight complexes, while HbpA-S is present as a monomer, indicating that the C-terminal region is required for formation of large aggregates. Growth studies showed that expression of HbpA-FL in the Δ mutant restored wild-type levels of growth in low-iron medium that contained Hb-Hp as the sole iron source, while HbpA-S failed to complement the Δ mutant. Protein localization studies in C. diphtheriae showed that HbpA-FL is present in both the supernatant and membrane fractions and that the C-terminal region is required for membrane anchoring. Purified HbpA-FL was able to enhance growth of the Δ mutant when added to culture medium that contained Hb-Hp as a sole iron source, suggesting that secreted HbpA is involved in the use of hemin iron from Hb-Hp. These studies extend our understanding of this novel Hb-Hp binding protein in this important human pathogen. Hemoproteins, such as Hb, are an abundant source of iron in humans and are proposed to be required by numerous pathogens to cause disease. In this report, we expand on our previous studies in further defining the role of HbpA in hemin iron acquisition in C. diphtheriae. HbpA is unique to C. diphtheriae and appears to function unlike any previously described bacterial iron-regulated Hb- or Hb-Hp-binding protein. HbpA is both secreted and present in the membrane and exists as a large aggregate that enhances its ability to bind Hb-Hp and promote hemin iron uptake. Current studies with HbpA will increase our understanding of iron transport systems in C. diphtheriae.
从血红蛋白-触珠蛋白 (Hb-Hp) 中获取亚铁血红素需要白喉棒状杆菌的铁调节表面蛋白 HtaA、ChtA 和 ChtC 以及最近鉴定的 Hb-Hp 结合蛋白 HbpA。我们之前表明，缺乏 C 端区域的纯化 HbpA 形式 (HbpA-S) 能够与 Hb-Hp 结合。在这项研究中，我们表明 HbpA 的 C 端区域显着增强了与 Hb-Hp 的结合。包含 C 端结构域的纯化 HbpA 形式（HbpA-FL）与 HbpA-S 相比，与 Hb-Hp 的结合要强得多。分子筛层析（SEC）表明 HbpA-FL 以及 HtaA-FL、ChtA-FL 和 ChtC-FL 以高分子量复合物的形式存在，而 HbpA-S 则以单体的形式存在，这表明 C 端区域是形成大聚集体所必需的。生长研究表明，在含有 Hb-Hp 作为唯一铁源的低铁培养基中，表达 HbpA-FL 的Δ突变体恢复了野生型的生长水平，而 HbpA-S 未能补充Δ突变体。白喉棒状杆菌中的蛋白定位研究表明，HbpA-FL 存在于上清液和膜部分中，并且 C 端区域是膜锚定所必需的。当添加到含有 Hb-Hp 作为唯一铁源的培养基中时，纯化的 HbpA-FL 能够增强Δ突变体的生长，这表明分泌的 HbpA 参与了从 Hb-Hp 中获取血红素铁的过程。这些研究扩展了我们对这种重要人类病原体中新型 Hb-Hp 结合蛋白的理解。血红素蛋白，如 Hb，是人类中丰富的铁源，被提议是许多病原体引起疾病所必需的。在本报告中，我们进一步扩展了我们之前的研究，以进一步定义 HbpA 在白喉棒状杆菌血红素铁获取中的作用。HbpA 是白喉棒状杆菌所特有的，其功能似乎与任何以前描述的细菌铁调节 Hb 或 Hb-Hp 结合蛋白都不同。HbpA 既分泌又存在于膜中，并且作为一个大的聚集体存在，增强了其与 Hb-Hp 结合的能力，并促进了血红素铁的摄取。目前对 HbpA 的研究将增加我们对白喉棒状杆菌中铁转运系统的理解。
