Roland K. Strong

Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron

Although iron is required to sustain life, its free concentration and metabolism have to be tightly regulated. This is achieved through a variety of iron-binding proteins including transferrin and ferritin. During infection, bacteria acquire much of their iron from the host by synthesizing siderophores that scavenge iron and transport it into the pathogen. We recently demonstrated that enterochelin, a bacterial catecholate siderophore, binds to the host protein lipocalin 2 (ref. 5). Here, we show that this event is pivotal in the innate immune response to bacterial infection. Upon encountering invading bacteria the Toll-like receptors on immune cells stimulate the transcription, translation and secretion of lipocalin 2; secreted lipocalin 2 then limits bacterial growth by sequestrating the iron-laden siderophore. Our finding represents a new component of the innate immune system and the acute phase response to infection.

The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.

First identified as a neutrophil granule component, neutrophil gelatinase-associated lipocalin (NGAL; also called human neutrophil lipocalin, 24p3, uterocalin, or neu-related lipocalin) is a member of the lipocalin family of binding proteins. Putative NGAL ligands, including neutrophil chemotactic agents such as N-formylated tripeptides, have all been refuted by recent biochemical and structural results. NGAL has subsequently been implicated in diverse cellular processes, but without a characterized ligand, the molecular basis of these functions remained mysterious. Here we report that NGAL tightly binds bacterial catecholate-type ferric siderophores through a cyclically permuted, hybrid electrostatic/cation-pi interaction and is a potent bacteriostatic agent in iron-limiting conditions. We therefore propose that NGAL participates in the antibacterial iron depletion strategy of the innate immune system.

An Iron Delivery Pathway Mediated by a Lipocalin

Despite the critical need for iron in many cellular reactions, deletion of the transferrin pathway does not block organogenesis, suggesting the presence of alternative methods to deliver iron. We show that a member of the lipocalin superfamily (24p3/Ngal) delivers iron to the cytoplasm where it activates or represses iron-responsive genes. Iron unloading depends on the cycling of 24p3/Ngal through acidic endosomes, but its pH sensitivity and its subcellular targeting differed from transferrin. Indeed, during the conversion of mesenchyme into epithelia (where we discovered the protein), 24p3/Ngal and transferrin were endocytosed by different cells that characterize different stages of development, and they triggered unique responses. These studies identify an iron delivery pathway active in development and cell physiology.

Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA.

The major histocompatibility complex (MHC) class I homolog, MICA, is a stress-inducible ligand for NKG2D, a C-type lectin–like activating immunoreceptor. The crystal structure of this ligand-receptor complex that we report here reveals an NKG2D homodimer bound to a MICA monomer in an interaction that is analogous to that seen in T cell receptor–MHC class I protein complexes. Similar surfaces on each NKG2D monomer interact with different surfaces on either the α1 or α2 domains of MICA. The binding interactions are large in area and highly complementary. The central section of the α2-domain helix, disordered in the structure of MICA alone, is ordered in the complex and forms part of the NKG2D interface. The extensive flexibility of the interdomain linker of MICA is shown by its altered conformation when crystallized alone or in complex with NKG2D.

Disulphide-isomerase-enabled shedding of tumour-associated NKG2D ligands

Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor—which are induced by genotoxic or cellular stress—trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal α3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.

The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake

We show here that megalin, a member of the low‐density lipoprotein receptor family expressed in polarized epithelia, binds NGAL with high affinity, as shown by surface plasmon resonance analysis. Furthermore, a rat yolk sac cell line known to express high levels of megalin, endocytosed NGAL by a mechanism completely blocked by an antibody against megalin.

Proof of principle for epitope-focused vaccine design

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

Lipocalin 2 promotes breast cancer progression

Here, we report that lipocalin 2 (Lcn2) promotes breast cancer progression, and we identify the mechanisms underlying this function. We first found that Lcn2 levels were consistently associated with invasive breast cancer in human tissue and urine samples. To investigate the function of Lcn2 in breast cancer progression, Lcn2 was overexpressed in human breast cancer cells and was found to up-regulate mesenchymal markers, including vimentin and fibronectin, down-regulate the epithelial marker E-cadherin, and significantly increase cell motility and invasiveness. These changes in marker expression and cell motility are hallmarks of an epithelial to mesenchymal transition (EMT). In contrast, Lcn2 silencing in aggressive breast cancer cells inhibited cell migration and the mesenchymal phenotype. Furthermore, reduced expression of estrogen receptor (ER) α and increased expression of the key EMT transcription factor Slug were observed with Lcn2 expression. Overexpression of ERα in Lcn2-expressing cells reversed the EMT and reduced Slug expression, suggesting that ERα negatively regulates Lcn2-induced EMT. Finally, orthotopic studies demonstrated that Lcn2-expressing breast tumors displayed a poorly differentiated phenotype and showed increased local tumor invasion and lymph node metastasis. Taken together, these in vitro, in vivo, and human studies demonstrate that Lcn2 promotes breast cancer progression by inducing EMT through the ERα/Slug axis and may be a useful biomarker of breast cancer.

The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2

Numerous bacteria cope with the scarcity of iron in their microenvironment by synthesizing small iron-scavenging molecules known as siderophores. Mammals have evolved countermeasures to block siderophore-mediated iron acquisition as part of their innate immune response. Secreted lipocalin 2 (Lcn2) sequesters the Escherichia coli siderophore enterobactin (Ent), preventing E. coli from acquiring iron and protecting mammals from infection by E. coli. Here, we show that the iroA gene cluster, found in many pathogenic strains of Gram-negative enteric bacteria, including E. coli, Salmonella spp., and Klebsiella pneumoniae, allows bacteria to evade sequestration of Ent by Lcn2. We demonstrate that C-glucosylated derivatives of Ent produced by iroA-encoded enzymes do not bind purified Lcn2, and an iroA-harboring strain of E. coli is insensitive to the growth inhibitory effects of Lcn2 in vitro. Furthermore, we show that mice rapidly succumb to infection by an iroA-harboring strain of E. coli but not its wild-type counterpart, and that this increased virulence depends on evasion of host Lcn2. Our findings indicate that the iroA gene cluster allows bacteria to evade this component of the innate immune system, rejuvenating their Ent-mediated iron-acquisition pathway and playing an important role in their virulence.

Iron Traffics in Circulation Bound to a Siderocalin (Ngal)-Catechol Complex

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal [known as Neutrophil Gelatinase Associated Lipocalin, Siderocalin, Lipocalin 2] sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography, and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH sensitive mechanism. Since catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal:catechol:iron complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal:siderophore interactions, but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.