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，宿主确实会主动限制组织和体液中的铁离子等必需营养物质。这种“营养免疫”确实有效地限制了细菌的繁殖，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，具有铁反应表面决定基因(ISD)。ISD系统包含细胞壁锚定蛋白、膜转运蛋白和胞浆蛋白，这些蛋白允许从血红蛋白中提取血红素，运输到细胞质中，并被降解以释放铁离子。因此，ISD系统是细菌病原体克服“营养免疫”的标志。我们研究了卢顿链霉菌的ISD系统，发现缺乏血红素特异性ABC转运蛋白(IsdEFL)的突变株不会失去利用血红素作为铁源的能力。我们鉴定了一种新的ABC转运蛋白(LHA)，缺乏该转运蛋白的突变株对作为铁源的血红素的生长能力显著降低。LHA属于ABC转运蛋白的能量耦合因子型(ECF)。ECF转运蛋白的底物特异性成分是高度疏水的膜蛋白，与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，只有满满一只手的配体是已知的。我们的数据表明，LHA是针对血红素的，它识别了一种新的底物，并使其成为第一个可能参与克服宿主先天免疫策略的ECF转运体。利用生化技术，我们将调查LHA是否具有预期的对血红素的高亲和力，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，表面锚定蛋白从血红素蛋白中提取血红素，并将其输送到膜转运体。这就提出了一个问题，为什么另外需要一个高亲和力的膜转运蛋白。为了解决这个问题，我们将调查LHA是否在功能上依赖于ISD系统，或者它是否是一个自主的血红素获取系统。我们假设LHA对血红素的亲和力足够高，足以从低丰度的血红素蛋白中提取血红素，如细胞色素或肌红蛋白。因此，它可能代表了一条不依赖于ISD的途径，以扩大血红蛋白底物的范围。为了挑战这一假说，我们将在生化水平上研究宿主血红素蛋白与LHA之间以及LHA与ISD蛋白之间的血红素转移。同时，我们将在替代宿主肉质葡萄球菌(缺乏其他ISD蛋白)中表达LHA系统，并在鲁顿葡萄球菌中产生等基因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.