革兰氏阴性菌血红素转运系统结构及功能特点

铁是大多数生物包括细菌生存的必需营养元素．对于感染宿主的致病细菌,血红素(heme/haem)可作为一种主要的铁来源．血红素转运系统在革兰氏阴性菌和革兰氏阳性菌中均有发现和鉴定,其转运机制在革兰氏阴性菌中有较为深入研究．革兰氏阴性菌血红素转运系统主要由分泌于细胞外的血红素载体(hemophore)、血红素受体、TonB ExbB ExbD复合物、ABC转运体、血红素降解蛋白和调控蛋白等结构单元组成．对参与该系统的各个蛋白结构特点以及它们之间的相互作用机制的讨论,有助于对病原菌致病机制的深入研究和抗菌新药的研发．     

细菌血红素降解蛋白结构特点及作用机制

铁离子是几乎所有生物包括细菌生存必需的营养元素. 在宿主体内,绝大多数的铁离子均以血红素的形式存在于各种血红素结合蛋白,如血红蛋白、肌红蛋白等. 当致病菌感染宿主后,血红素将成为某些致病菌主要的铁离子来源. 致病菌编码血红素转运系统,并利用该系统将血红素转运至胞浆,在胞浆血红素被细菌的血红素降解蛋白降解,释放铁离子供细菌利用. 在致病菌中,目前至少有两种血红素降解酶被发现和鉴定. 第一种为经典的血红素氧化酶(heme oxygenase, HO),它催化血红素氧化形成胆绿素、一氧化碳(CO)和Fe2+;第二种非经典降解酶,包括金黄色葡萄球菌的IsdG/IsdI蛋白及其同系物MhuD蛋白,催化血红素分别产生staphylobilin和mycobilin. 另外,部分细菌内存在其它血红素降解因子,其与前两种血红素降解酶无结构同源性,但在血红素降解实验中可产生胆绿素(biliverdin)或CO,因而被鉴定为“血红素降解蛋白”. 对细菌血红素降解蛋白分子结构解析及作用机制的深入理解,将有助于新的血红素降解蛋白的发现和鉴定.     

部分革兰氏阴性菌TonB蛋白的结构特点及作用机制

摘要:在革兰氏阴性菌内,TonB系统对环境中的重要营养物质的摄取至关重要。TonB系统由锚定在内膜的ExbB-ExbD和周质蛋白TonB组成,它为TonB依赖性外膜受体(TBDTs)提供能量,使其转运营养物质。TonB系统普遍参与了铁、血红素、维生素B12、碳水化合物及多种过渡金属元素等多种重要物质的转运过程。TonB蛋白的功能与其特殊的结构密切相关,它的结构包括起固定作用的氨基端结构域、柔韧可变的脯氨酸富集的中间结构域和与TonB依赖性受体相互作用的羧基端结构域。虽然TonB蛋白结构特点较为清晰,但 其精确作用机制尚未被完全揭示。本文综述了革兰氏阴性菌TonB依赖性的营养物质摄取、TonB蛋白的结构特点、作用机制模型及表达调控,以期为进一步研究TonB蛋白功能提供理论基础和参考。     

革兰氏阴性菌脂蛋白Lol系统转运蛋白的功能及表面展示分泌机制

细菌脂蛋白是细胞膜的重要组成成分,在革兰氏阴性菌的生理及致病性中扮演着重要的角色。革兰氏阴性菌中已知负责胞内脂蛋白转运的是Lol(Localization of lipoprotein)系统。该系统识别成熟脂蛋白的分泌信号,将外膜脂蛋白转运并定位于细胞外膜内侧。近年来的研究发现,跨细胞外膜进行表面展示的脂蛋白实际上在革兰氏阴性菌中广泛存在,其分泌机制开始成为研究热点。为了对革兰氏阴性菌中脂蛋白分泌机制的研究现状有一个系统全面的了解,本文概述了脂蛋白转运过程中Lol系统5个转运蛋白的功能与保守性、不同细菌中脂蛋白分泌信号的差异以及表面展示脂蛋白可能的分泌机制。     

革兰氏阴性菌亚铁离子转运系统的组成及作用机制

几乎所有细菌的生长都离不开铁元素。在有氧的环境中,三价铁离子几乎无法被细菌直接利用。但是在宿主胃肠道中,铁元素主要以可溶性的亚铁离子形式存在,它们可通过革兰氏阴性菌外膜直接进入胞周质,在周质通过亚铁离子转运系统,将铁离子转运至胞浆供细菌利用。绝大多数阴性菌主要是通过Feo转运系统利用亚铁离子,大肠杆菌的Feo转运系统由feoA、feoB和feoC3个基因组成。除Feo转运系统外,还发现Yfe转运系统、Efe转运系统、Sit转运系统等。本文重点介绍革兰氏阴性菌Feo转运系统的组成及作用机制,以期为进一步研究细菌亚铁离子的转运机制提供参考。     

革兰氏阴性菌TonB依赖性受体的功能与结构研究进展*

为维持生长所需,革兰氏阴性菌需要从外界摄取多种营养物质。分子量小于600 Da的分子可以通过自由扩散的方式通过革兰氏阴性菌的外膜,而大分子物质则需要特殊的转运系统才能将其转运至革兰氏阴性菌的胞内。革兰氏阴性菌对大分子营养物质的识别和转运主要由TonB依赖性受体负责完成。所有革兰氏阴性菌中均有TonB依赖性受体的存在,然而不同种类的革兰氏阴性菌拥有TonB依赖性受体的数量不同且功能各异。最近研究表明,TonB依赖性受体不仅参与了铁、血红素、锰、锌、镍、维生素、碳水化合物等多种营养物质的摄取,而且参与了蛋白酶的分泌。为对TonB依赖性受体提供更为深入和系统的理解,详细介绍了目前已知的TonB依赖性受体的功能及结构,以期为更进一步探知TonB依赖性受体未知功能提供可参考依据。     

部分革兰氏阴性菌脂多糖运输蛋白LptD的结构及功能研究进展

在革兰氏阴性菌中,脂多糖是外膜的重要组成部分,并参与构成细菌的固有免疫。而在大多数革兰氏阴性菌中,Lpt系统都是运输脂多糖的唯一途径,在该系统中LptD作为一个跨膜的外膜蛋白,也是脂多糖输出的最后一步,因此被许多学者称作脂多糖运输的"命门"。LptD参与多种重要的生物学功能,包括有机溶剂耐受性、疏水性抗生素耐受性、膜通透性等。但近来的研究表明,LptD最重要的功能是参与了脂多糖的运输,也因为其参与脂多糖运输而具有了多种功能。本文重点介绍部分革兰氏阴性菌LptD的蛋白结构及其功能研究进程,以期为进一步研究其它革兰氏阴性菌脂多糖运输通路(Lpt通路)及该通路上各蛋白间的相互作用机制提供参考。     

革兰氏阴性菌TonB-ExbB-ExbD复合物的功能、作用机制、分布及进化

革兰氏阴性菌在生长繁殖过程中需要从外界摄取营养物质。一些小分子营养物质可以自由地通过革兰氏阴性菌的细胞膜,而一些大分子营养物质的转运需要特异性的TonB复合物依赖性的外膜受体进行转运。TonB复合物由TonB、ExbB、ExbD构成,是革兰氏阴性菌对外界营养物质主动转运过程的能量提供单位,在革兰氏阴性菌分布广泛。近年来,对TonB-ExbB-ExbD复合物的功能、结构及作用机制取得了重大研究进展,然而此复合物在不同的细菌也存在功能及作用机制上的差异。基于此背景,本文综述了TonB复合物的功能和结构研究进展,并分析了TonB复合物在革兰氏阴性菌中的分布、进化,比较了不同革兰氏阴性菌此复合物的差异,有助于进一步发现和揭示TonB复合物的新功能与作用机制。     

革兰氏阴性菌脂多糖运输系统的构成及作用机制

革兰氏阴性菌包含有两层组分不同的膜结构——内膜和外膜,对大多数革兰氏阴性菌而言,脂多糖(lipopolysaccharides,LPS)是其外膜上最主要的脂质成分,锚定在外膜小叶(the outer leaflet of the OM)上,是革兰氏阴性菌固有免疫的重要组成部分。脂多糖运输系统(lipopolysaccharide transport system,Lpt)将胞内装配完整的LPS正确装配到外膜,使得与脂多糖相关的阻渗、有机溶剂耐受性、疏水性抗生素耐受性、膜通透性等功能得以实现。该运输系统的正确作用主要依赖7个不同的脂多糖运输蛋白(Lpt ABCDEFG)协同完成,整个系统贯穿细菌内膜至外膜,由内膜上ABC转运体复合物Lpt B2FG、胞质内转运协同蛋白Lpt A/C及被许多学者称作脂多糖运输的"命门"的外膜蛋白复合物Lpt DE共同构成。本文就革兰氏阴性菌脂多糖的具体结构功能进行简介,进而综述脂多糖运输系统的7个蛋白的构成和作用机制,以期为进一步研究该系统中每个蛋白的功能提供理论基础及参考。     

革兰氏阴性菌TonB-ExbB-ExbD复合物的功能、作用机制、分布及进化分析

革兰氏阴性菌在生长繁殖过程中需要从外界摄取营养物质。一些小分子营养物质可以自由地通过革兰氏阴性菌的细胞膜,而一些大分子营养物质的转运需要特异性的TonB复合物依赖性的外膜受体进行转运。TonB复合物由TonB、ExbB、ExbD构成,是革兰氏阴性菌对外界营养物质主动转运过程的能量提供单位,在革兰氏阴性菌分布广泛。近年来,对TonB-ExbB-ExbD复合物的功能、结构及作用机制取得了重大研究进展,然而此复合物在不同的细菌也存在功能及作用机制上的差异。基于此背景,本文综述了TonB复合物的功能和结构研究进展,并分析了TonB复合物在革兰氏阴性菌中的分布、进化,比较了不同革兰氏阴性菌此复合物的差异,有助于进一步发现和揭示TonB复合物的新功能。     

Free and hemophore-bound heme acquisitions through the outer membrane receptor HasR have different requirements for the TonB-ExbB-ExbD complex

Many gram-negative bacteria have specific outer membrane receptors for free heme, hemoproteins, and hemophores. Heme is a major iron source and is taken up intact, whereas hemoproteins and hemophores are not transported: the iron-containing molecule has to be stripped off at the cell surface, with only the heme moiety being taken up. The Serratia marcescens hemophore-specific outer membrane receptor HasR can transport either heme itself or heme bound to the hemophore HasA. This second mechanism is much more efficient and requires a higher TonB-ExbB-ExbD (TonB complex) concentration than does free or hemoglobin-bound heme uptake. This requirement for more of the TonB complex is associated with a higher energy requirement. Indeed, the sensitivity of heme-hemophore uptake to the protonophore carbonyl cyanide m-chlorophenyl hydrazone is higher than that of heme uptake from hemoglobin. We show that a higher TonB complex concentration is required for hemophore dissociation from the receptor. This dissociation is concomitant with heme uptake. We propose that increasing the TonB complex concentration drives more energy to the outer membrane receptor and speeds up the release of empty hemophores, which, if they remained on receptors, would inhibit heme transport.     

Structural biology of bacterial iron uptake

To fulfill their nutritional requirement for iron, bacteria utilize various iron sources which include the host proteins transferrin and lactoferrin, heme, and low molecular weight iron chelators termed siderophores. The iron sources are transported into the Gram-negative bacterial cell via specific uptake pathways which include an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. Over the past two decades, structures for the proteins involved in bacterial iron uptake have not only been solved, but their functions have begun to be understood at the molecular level. However, the elucidation of the three dimensional structures of all components of the iron uptake pathways is currently limited. Despite the low sequence homology between different bacterial species, the available three-dimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.     

Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores

The major mechanisms by which Gram-negative bacteria acquire heme from host heme-carrier proteins involve either direct binding to specific outer membrane receptors or release of bacterial hemophores that take up heme from host heme carriers and shuttle it back to specific receptors. The ability to interact with and remove heme from carrier proteins distinguishes heme from conceptually similar siderophore and vitamin B12 receptors. Recent genetic, biochemical and crystallization studies have started to unravel the mechanism and molecular interactions between heme-carrier proteins and components of bacterial heme assimilation systems.     

Comparative analysis of structural and dynamic properties of the loaded and unloaded hemophore HasA: functional implications

A heme-acquisition system present in several Gram-negative bacteria requires the secretion of hemophores. These extracellular carrier proteins capture heme and deliver it to specific outer membrane receptors. The Serratia marcescens HasA hemophore is a monodomain protein that binds heme with a very high affinity. Its α/β structure, as that of its binding pocket, has no common features with other iron- or heme-binding proteins. Heme is held by two loops L1 and L2 and coordinated to iron by an unusual ligand pair, H32/Y75. Two independent regions of the hemophore β-sheet are involved in HasA-HasR receptor interaction. Here, we report the 3-D NMR structure of apoHasA and the backbone dynamics of both loaded and unloaded hemophore. While the overall structure of HasA is very similar in the apo and holo forms, the hemophore presents a transition from an open to a closed form upon ligand binding, through a large movement, of up to 30 Å, of loop L1 bearing H32. Comparison of loaded and unloaded HasA dynamics on different time scales reveals striking flexibility changes in the binding pocket. We propose a mechanism by which these structural and dynamic features provide the dual function of heme binding and release to the HasR receptor.     

Heme acquisition by hemophores

Bacterial hemophores are secreted to the extracellular medium, where they scavenge heme from various hemoproteins due to their higher affinity for this compound, and return it to their specific outer membrane receptor. HasR, the outer membrane receptor of the HasA hemophore, assumes multiple functions which require various energy levels. Binding of heme and, of heme-free or heme-loaded hemophores is energy-independent. Heme transfer from the holo-hemophore to the outer membrane receptor is also energy-independent. In contrast, heme transport and hemophore release require basal or high levels of TonB and proton motive force, respectively. In addition, HasR is a component of a signaling cascade, regulating expression of the has operon via specific sigma and anti-sigma factors encoded by genes clustered at the has operon. The signal is the heme landing on HasR in the presence of the hemophore in its apo form. The has system is the only system thus far characterized in which the anti-sigma factor is submitted to the same signaling cascade as the target operon. Specific autoregulation of the has system, combined with negative regulation by the Fur protein, permits bacterial adaptation to the available iron source. In the presence of a heme-loaded hemophore, inactive anti-sigma factor is accumulated and can be activated as soon as the heme source dries up. Hence, the has system, instead of being submitted to amplification like other systems regulated by sigma anti-sigma factors, functions by pulses triggered by heme availability.     

Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB

TonB is a cytoplasmic membrane protein required for active transport of various essential substrates such as heme and iron siderophores through the outer membrane receptors of Gram-negative bacteria. This protein spans the periplasm, contacts outer membrane transporters by its C-terminal domain, and transduces energy from the protonmotive force to the transporters. The TonB box, a relatively conserved sequence localized on the periplasmic side of the transporters, has been shown to directly contact TonB.While Serratia marcescens TonB functions with various transporters, HasB, a TonB-like protein, is dedicated to the HasR transporter. HasR acquires heme either freely or via an extracellular heme carrier, the hemophore HasA, that binds to HasR and delivers heme to the transporter. Here, we study the interaction of HasR with a HasB C-terminal domain and compare it with that obtained with a TonB C-terminal fragment. Analysis of the thermodynamic parameters reveals that the interaction mode of HasR with HasB differs from that with TonB, the difference explaining the functional specificity of HasB for HasR. We also demonstrate that the presence of the substrate on the extracellular face of the transporter modifies, via enthalpy-entropy compensation, the interaction with HasB on the periplasmic face. The transmitted signal depends on the nature of the substrate. While the presence of heme on the transporter modifies only slightly the nature of interactions involved between HasR and HasB, hemophore binding on the transporter dramatically changes the interactions and seems to locally stabilize some structural motifs. In both cases, the HasR TonB box is the target for those modifications.     

Mechanisms and regulation of iron and heme utilization in Gram-negative bacteria

Iron and heme are essential nutrients for most pathogenic microorganisms and play a pivotal role in microbial pathogenesis. To survive within the iron-limited environment of the host, bacteria utilize iron-siderophore complexes, iron-binding proteins (transferrin, lactoferrin), free heme and heme bound to hemoproteins (hemoglobin, haptoglobin, hemopexin). A mechanism of iron and heme transport depends on the structures of Gram-negative bacterial membranes. Siderophores, hemophores and outer membrane receptors take part in iron or heme binding. The transport of these ligands across the outer membrane involves outer membrane receptors. The energy for this transport is delivered from the inner membrane by a TonB-ExbB-ExbD complex. The transport across the cytoplasmic membrane involves periplasmic and inner membrane proteins comprising the ABC systems, which utilize the energy derived from ATP hydrolysis. The major regulatory role in iron homeostasis plays a Fur-Fe2+ repressor.     

Differential Contributions of the Outer Membrane Receptors PhuR and HasR to Heme Acquisition in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 encodes two outer membrane receptors, PhuR (Pseudomonas heme uptake) and HasR (heme assimilation system). The HasR and PhuR receptors have distinct heme coordinating ligands and substrate specificities. HasR is encoded in an operon with a secreted hemophore, HasAp. In contrast the non-hemophore-dependent PhuR is encoded within an operon along with proteins required for heme translocation into the cytoplasm. Herein we report on the contributions of the HasR and PhuR receptors to heme uptake and utilization. Employing bacterial genetics and isotopic [¹³C]heme labeling studies we have shown both PhuR and HasR are required for optimal heme utilization. However, the unique His-Tyr-ligated PhuR plays a major role in the acquisition of heme. In contrast the HasR receptor plays a primary role in the sensing of extracellular heme and a supplementary role in heme uptake. We propose PhuR and HasR represent non-redundant heme receptors, capable of accessing heme across a wide range of physiological conditions on colonization of the host.