A novel ECF-type transporter promotes heme acquisition in Staphylococcus lugdunensis to overcome nutritional limitation

在病原体侵袭性感染过程中，宿主确实对组织和体液中的必需营养物质如铁离子实施了主动限制。这种“营养免疫”确实有效地限制了细菌的增殖，是先天免疫系统的重要组成部分。细菌病原体如金黄色葡萄球菌和卢顿葡萄球菌具有铁响应表面决定因子（isd）位点。Isd系统包含细胞壁锚定蛋白，膜转运蛋白和细胞质蛋白，这些蛋白允许血红素从血红蛋白中提取，运输到细胞质中，并降解以释放铁离子。因此，Isd系统是细菌病原体能够克服“营养免疫”的标志。我们研究了S. lugdunensis的Isd系统，发现缺乏血红素特异性ABC转运体（IsdEFL）的突变体并没有失去使用血红素作为铁源的能力。我们发现了一种新的ABC转运蛋白（Lha），缺乏这种转运蛋白的突变体在血红素作为铁源上的生长能力大大降低。Lha属于ABC转运体的能量耦合因子型（ECF）。ECF转运体的底物特异性成分是高度疏水的膜蛋白，对底物具有非凡的亲和力。ECF转运体代表微量营养素摄取系统，目前已知的配体只有一小部分。我们的数据表明，Lha对血红素具有特异性，鉴定出一种新的底物，并使其成为第一个被认为参与克服宿主先天免疫策略的ECF转运蛋白。利用生化技术，我们将研究Lha是否对血红素具有预期的高亲和力，以及它是否优于传统的血红素特异性脂蛋白IsdE。在传统的Isd系统中，表面固定蛋白从血红蛋白中提取血红素，并将其输送到膜转运蛋白中。这就提出了一个问题，为什么还需要高亲和力的膜转运蛋白。为了解决这个问题，我们将研究Lha是否在功能上依赖于Isd系统，或者它是否是一个自主血红素获取系统。我们假设Lha对血红素的亲和力足够高，可以从低丰度的血红蛋白（如细胞色素或肌红蛋白）中提取血红素。因此，它可以代表一个独立于isd的途径来扩大血红蛋白底物范围。为了挑战这一假设，我们将在生化水平上研究宿主血红蛋白和Lha之间以及Lha和Isd蛋白之间的血红素转移。同时，我们将在代理宿主肉葡萄球菌（Staphylococcus carnosus）中表达Lha系统（缺乏其他Isd蛋白），并在S. lugdunensis中创建等基因Isd缺失突变。这将允许在生理水平上确定Lha是否在功能上依赖于Isd蛋白。

During invasive infection of pathogens, the host does implement active limitation of essential nutrients such as iron ions within the tissues and body fluids. This "nutritional immunity" does effectively limit bacterial proliferation and is an important part of the innate immune system. Bacterial pathogens such as the staphylococcus species S. aureus and S. lugdunensis possess iron-responsive surface determinant (isd) loci. Isd systems contain cell wall-anchored proteins, a membrane transporter and cytosolic proteins allowing heme to be extracted from hemoglobin, to be transported into the cytoplasm, and to be degraded to release the iron ion. As such, Isd systems are hallmarks of bacterial pathogens allowing to overcome “nutritional immunity”.We investigated the Isd system of S. lugdunensis and found that a mutant lacking the heme specific ABC transporter (IsdEFL) did not lose its ability to use heme as an iron source. We identified a novel ABC transporter (Lha) and mutants lacking this transporter showed a strongly reduced ability to thrive on heme as an iron source. Lha belongs to the energy coupling factor-type (ECF) of ABC transporters. The substrate-specific components of ECF transporters are highly hydrophobic membrane proteins with extraordinary affinity towards their substrate. ECF transporters represent micronutrient uptake systems and only a hand full of ligands are known. Our data indicate that Lha is specific for heme, identifying a novel substrate and making it the first ECF transporter putatively involved in overcoming host innate immune strategies. Using biochemical techniques, we will investigate whether Lha possesses the expected high affinity to heme and whether it outperforms the conventional heme-specific lipoprotein IsdE. In a conventional Isd system, surface-anchored proteins extract heme from hemoproteins and funnel it to the membrane transporter. This raises the question why a high-affinity membrane transporter is needed in addition. To address this, we will investigate whether Lha is functionally dependent on the Isd system or whether it is an autonomous heme acquisition system. We hypothesize that the affinity of Lha towards heme is high enough to extract heme from low-abundance hemoproteins such as cytochromes or myoglobin. Thereby it could represent an Isd-independent pathway to expand the hemoprotein substrate range. To challenge this hypothesis we will investigate heme transfer between host hemoproteins and Lha as well as between LhaS and the Isd proteins on the biochemical level. Simultaneously, we will express the Lha system in the surrogate host Staphylococcus carnosus (lacking other Isd proteins) and create isogenic isd deletion mutations in S. lugdunensis. This will allow identifying whether Lha is functionally dependent on the Isd proteins on the physiological level.