PDF(Pubmed)
Abstract：
In the previous study, it was shown that Riemerella anatipestifer (R. anatipestifer, RA), a pathogen in ducks and some other birds, encodes a hemin uptake system. The R. anatipestifer hemin uptake receptor RhuR is a TonB2-dependent hemin transporter. However, it remains unclear whether R. anatipestifer encodes additional TonB-dependent hemin transporters. Herein, we demonstrated that R. anatipestifer hemin uptake receptor B (RhuB) of R. anatipestifer CH-1 (RA CH-1) was negatively regulated by iron and mediated by the Fur protein, and knocking out rhuB damaged the ability of RA CH-1 to utilize iron from duck hemoglobin (Hb) but not that from duck serum. Moreover, the ability to use iron from Hb was restored by the expression rhuB in trans. Furthermore, the RhuB of RA CH-1 is a membrane protein, and recombinant RhuB could bind hemin at a 1:1 molar ratio in vitro. Compared to that of ΔtonB1ΔrhuR, the ability of ΔtonB1ΔrhuRΔrhuB to utilize hemin was impaired; meanwhile, compared to that of ΔtonB2ΔrhuR, the hemin utilization ability of ΔtonB2ΔrhuRΔrhuB was not affected, indicating that RhuB is a TonB2-dependent receptor. Compared to ΔrhuB, ΔrhuBΔrhuA did not affect hemin utilization. However, compared to ΔrhuA, ΔrhuBΔrhuA had reduced ability to utilize hemin, suggesting that RhuA relies on RhuB for its activity. Finally, the deletion of rhuB did not affect the virulence of RA CH-1. These results suggested that RhuB encodes a TonB2-dependent hemin receptor. The characterization of the second TonB-dependent receptor in R. anatipestifer enriches our understanding of the hemin uptake system of this bacterium.IMPORTANCEIron is essential for the survival of most bacteria, and hemin of hemoglobin can serve as an important iron source. In our previous studies, we showed that R. anatipestifer CH-1 encodes a TonB2-dependent hemin receptor RhuR, which is involved in hemin uptake. The deletion of rhuR did not abolish hemin utilization by RA CH-1. We hypothesized that additional hemin uptake systems exist in this bacterium. In this study, we identified the second TonB2-dependent hemin receptor RhuB in RA CH-1 through hemin utilization, protein localization, and hemin-binding experiments. The duck infection model showed that the deletion of rhuB did not affect the virulence of RA CH-1. This study is not only important for further understanding the hemin utilization mechanism of R. anatipestifer, but also for enriching the hemin uptake transporters of gram-negative bacteria.
摘要：
在之前的研究中,研究表明，Riemerellaanatipestifer(R.anatipestifer,RA),鸭子和其他一些鸟类的病原体，编码血红素摄取系统。R.anatipestifer血红素摄取受体RhuR是TonB2依赖性血红素转运蛋白。然而,目前尚不清楚R.anatipestifer是否编码额外的TonB依赖性血红素转运蛋白。在这里，我们证明了R.anatipestiferCH-1（RACH-1）的血红素摄取受体B（RhuB）受铁的负调控，并由Fur蛋白介导，敲除rhuB会损害RACH-1利用鸭血红蛋白（Hb）中铁的能力，但不会损害鸭血清中的铁。此外，通过反式表达rhuB恢复了从Hb中利用铁的能力。此外，RACH-1的RhuB是一种膜蛋白，重组RhuB可以在体外以1:1的摩尔比结合血红素。与ΔtonB1ΔrhuR相比，ΔtonB1ΔrhuRΔrhuB利用血红素的能力受损；同时，与ΔtonB2ΔrhuR相比，ΔtonB2ΔrhuRΔrhuB的血红素利用能力不受影响，表明RhuB是TonB2依赖性受体。与ΔrhuB相比，ΔrhuBΔrhuA不影响血红素的利用。然而,与ΔrhuA相比，ΔrhuBΔrhuA降低了利用血红素的能力，这表明RhuA的活动依赖于RhuB。最后,rhuB的缺失不影响RACH-1的毒力。这些结果表明RhuB编码TonB2依赖性血红素受体。R.anatipestifer中第二个TonB依赖性受体的表征丰富了我们对该细菌的血红素摄取系统的理解。重要铁对于大多数细菌的生存至关重要，血红蛋白中的血红素可以作为重要的铁源。在我们之前的研究中,我们显示R.anatipestiferCH-1编码TonB2依赖性血红素受体RhuR，这与血红素摄取有关。rhuR的缺失并没有消除RACH-1对血红素的利用。我们假设该细菌中存在额外的血红素摄取系统。在这项研究中,我们通过利用血红素鉴定了RACH-1中第二个TonB2依赖性血红素受体RhuB，蛋白质定位，和血红素结合实验。鸭感染模型显示，rhuB的缺失不影响RACH-1的毒力。本研究不仅对进一步了解鼠疫菌的血红素利用机制,还用于富集革兰阴性细菌的血红素摄取转运蛋白。
