Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
Research Center of Avian Disease, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
Appl Environ Microbiol. 2021 Jul 13;87(15):e0036721. doi: 10.1128/AEM.00367-21.
Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer, a Gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes hemoglobin-derived hemin as an iron source; however, the mechanism by which this bacterium acquires hemin from hemoglobin is largely unknown. Here, disruption was shown to impair iron utilization from duck hemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane hemin-binding protein, but it could not extract hemin from duck hemoglobin. Mutagenesis studies showed that recombinant RhuA, RhuA, and RhuA lost hemin-binding ability, suggesting that amino acid sites at tyrosine 144 (Y144), Y177, and histidine 149 (H149) are crucial for hemin binding. Furthermore, , the gene adjacent to , encodes a TonB2-dependent hemin transporter. The function of in duck hemoglobin utilization was abolished in the mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1 Δ rather than R. anatipestifer CH-1 Δ Δ, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA hemin utilization locus exhibits no similarity to those of characterized hemin transport systems. Thus, this locus is a novel hemin uptake locus with homologues distributed mainly in the phylum. In vertebrates, hemin from hemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain hemin from hemoglobin, but the mechanisms of hemin acquisition from hemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of hemin acquisition from mammalian hemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in hemin acquisition from duck hemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane hemin-binding protein, and RhuR was identified as a TonB2-dependent hemin transporter. Moreover, the function of RhuA in hemoglobin utilization is RhuR dependent and not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel hemin transportation system of Gram-negative bacteria. This study not only was important for understanding hemin uptake in R. anatipestifer but also enriched the knowledge about the hemin transportation pathway in Gram-negative bacteria.
铁是大多数细菌复制所必需的元素，包括鸭疫里默氏菌，一种鸭和其他鸟类的革兰氏阴性细菌病原体。鸭疫里默氏菌利用血红蛋白衍生的血红素作为铁源；然而，该细菌从血红蛋白中获取血红素的机制在很大程度上尚不清楚。在这里， 破坏显示可损害鸭疫里默氏菌 CH-1 中从鸭血红蛋白中利用铁。此外，鉴定出假定的脂蛋白 RhuA 是一种表面暴露的外膜血红素结合蛋白，但它不能从鸭血红蛋白中提取血红素。诱变研究表明，重组 RhuA、RhuA 和 RhuA 失去了血红素结合能力，表明酪氨酸 144（Y144）、Y177 和组氨酸 149（H149）处的氨基酸位点对于血红素结合至关重要。此外， ，位于 基因的相邻基因编码一种 TonB2 依赖性血红素转运蛋白。在 突变株中， 基因在鸭血红蛋白利用中的功能被废除，重组 RhuA 能够结合鸭疫里默氏菌 CH-1 Δ 的细胞表面，而不是鸭疫里默氏菌 CH-1 Δ Δ ，表明 RhuA 与 RhuR 结合发挥作用。RhuR-RhuA 血红素利用基因座的序列与已鉴定的血红素转运系统没有相似性。因此，该基因座是一种新型的血红素摄取基因座，其同源物主要分布在 门。 在脊椎动物中，血红蛋白中的血红素是感染性细菌的重要铁源。许多细菌可以从血红蛋白中获得血红素，但细菌从血红蛋白中获取血红素的机制不同。此外，大多数研究都集中在从哺乳动物血红蛋白中获取血红素的机制上。在这项研究中，我们发现鸭疫里默氏菌 CH-1 的 RhuR-RhuA 基因座，一种鸭病原体，通过独特的途径参与从鸭血红蛋白中获取血红素。鉴定出 RhuA 是一种暴露的外膜血红素结合蛋白，RhuR 是一种 TonB2 依赖性血红素转运蛋白。此外，RhuA 在血红蛋白利用中的功能依赖于 RhuR，反之则不然。RhuR 和 RhuA 的同源物广泛分布于海洋环境、动物和植物中的细菌中，代表了革兰氏阴性细菌的一种新型血红素运输系统。这项研究不仅对理解鸭疫里默氏菌中的血红素摄取很重要，而且丰富了革兰氏阴性细菌中血红素运输途径的知识。
