Adhikari P, Kirby S D, Nowalk A J, Veraldi K L, Schryvers A B, Mietzner T A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
J Biol Chem. 1995 Oct 20;270(42):25142-9. doi: 10.1074/jbc.270.42.25142.
Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram-negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic iron-binding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC). In this study we describe the cloning, overexpression, and purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2'-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.
细菌铁转运对于病原体在宿主环境中的生长至关重要，在宿主环境中，铁作为一种非特异性免疫形式含量有限。对于流感嗜血杆菌等革兰氏阴性菌而言，铁首先必须穿过外膜进入周质空间，然后从周质转运至胞质溶胶。流感嗜血杆菌表达一种由hitA基因编码的周质铁结合蛋白。该基因是一个三基因操纵子中的第一个基因，该操纵子据称编码一个经典的高亲和力铁获取系统，包括hitA、一种细胞质通透酶（hitB）和一种核苷酸结合蛋白（hitC）。在本研究中，我们描述了流感嗜血杆菌hitA基因产物的克隆、过表达和纯化。通过证明其在体外以及在铁载体缺陷型大肠杆菌的周质空间内与结合在化学铁螯合剂2,2'-联吡啶上的铁竞争的能力，明确确定了该蛋白的功能。最后，通过将这种铁载体缺陷型大肠杆菌互补以使其能在含联吡啶的培养基上生长，证明了功能性hitABC操纵子对于铁获取的重要性。这些研究代表了对一种主动运输系统的详细遗传、生化和生理学描述，该系统经过进化可有效转运铁，因此在革兰氏阴性致病细菌中广泛分布。
