Marcus Miethke

Siderophore-Based Iron Acquisition and Pathogen Control

SUMMARY High-affinity iron acquisition is mediated by siderophore-dependent pathways in the majority of pathogenic and nonpathogenic bacteria and fungi. Considerable progress has been made in characterizing and understanding mechanisms of siderophore synthesis, secretion, iron scavenging, and siderophore-delivered iron uptake and its release. The regulation of siderophore pathways reveals multilayer networks at the transcriptional and posttranscriptional levels. Due to the key role of many siderophores during virulence, coevolution led to sophisticated strategies of siderophore neutralization by mammals and (re)utilization by bacterial pathogens. Surprisingly, hosts also developed essential siderophore-based iron delivery and cell conversion pathways, which are of interest for diagnostic and therapeutic studies. In the last decades, natural and synthetic compounds have gained attention as potential therapeutics for iron-dependent treatment of infections and further diseases. Promising results for pathogen inhibition were obtained with various siderophore-antibiotic conjugates acting as “Trojan horse” toxins and siderophore pathway inhibitors. In this article, general aspects of siderophore-mediated iron acquisition, recent findings regarding iron-related pathogen-host interactions, and current strategies for iron-dependent pathogen control will be reviewed. Further concepts including the inhibition of novel siderophore pathway targets are discussed.

Copper Stress Affects Iron Homeostasis by Destabilizing Iron-Sulfur Cluster Formation in Bacillus subtilis

Copper and iron are essential elements for cellular growth. Although bacteria have to overcome limitations of these metals by affine and selective uptake, excessive amounts of both metals are toxic for the cells. Here we investigated the influences of copper stress on iron homeostasis in Bacillus subtilis, and we present evidence that copper excess leads to imbalances of intracellular iron metabolism by disturbing assembly of iron-sulfur cofactors. Connections between copper and iron homeostasis were initially observed in microarray studies showing upregulation of Fur-dependent genes under conditions of copper excess. This effect was found to be relieved in a csoR mutant showing constitutive copper efflux. In contrast, stronger Fur-dependent gene induction was found in a copper efflux-deficient copA mutant. A significant induction of the PerR regulon was not observed under copper stress, indicating that oxidative stress did not play a major role under these conditions. Intracellular iron and copper quantification revealed that the total iron content was stable during different states of copper excess or efflux and hence that global iron limitation did not account for copper-dependent Fur derepression. Strikingly, the microarray data for copper stress revealed a broad effect on the expression of genes coding for iron-sulfur cluster biogenesis (suf genes) and associated pathways such as cysteine biosynthesis and genes coding for iron-sulfur cluster proteins. Since these effects suggested an interaction of copper and iron-sulfur cluster maturation, a mutant with a conditional mutation of sufU, encoding the essential iron-sulfur scaffold protein in B. subtilis, was assayed for copper sensitivity, and its growth was found to be highly susceptible to copper stress. Further, different intracellular levels of SufU were found to influence the strength of Fur-dependent gene expression. By investigating the influence of copper on cluster-loaded SufU in vitro, Cu(I) was found to destabilize the scaffolded cluster at submicromolar concentrations. Thus, by interfering with iron-sulfur cluster formation, copper stress leads to enhanced expression of cluster scaffold and target proteins as well as iron and sulfur acquisition pathways, suggesting a possible feedback strategy to reestablish cluster biogenesis.

Ferri-bacillibactin uptake and hydrolysis in Bacillus subtilis.

Upon iron limitation, Bacillus subtilis secretes the catecholic trilactone (2,3‐dihydroxybenzoate‐glycine‐threonine)3 siderophore bacillibactin (BB) for ferric iron scavenging. Here, we show that ferri‐BB uptake is mediated by the FeuABC transporter and that YuiI, a novel trilactone hydrolase, catalyses ferri‐BB hydrolysis leading to cytosolic iron release. Among several Fur‐regulated ABC transport mutants, only ΔfeuABC exhibited impaired growth during iron starvation. Quantification of intra‐ and extracellular (ferri)‐BB in iron‐depleted ΔfeuABC cultures revealed a fourfold increase of the extracellular siderophore concentration, confirming a blocked ferri‐BB uptake in the absence of FeuABC. Ferri‐BB was found to bind selectively to the periplasmic binding protein FeuA (Kd = 57 ± 1 nM), proving high‐affinity transport of the iron‐charged siderophore. During iron starvation, a ΔyuiI mutant displayed impaired growth and strong intracellular (30‐fold) and extracellular (6.5‐fold) (ferri)‐BB accumulation. Kinetic studies in vitro revealed that YuiI hydrolyses both BB and ferri‐BB. While BB hydrolysis led to strong accumulation of the tri‐ and dimeric reaction intermediates, ferri‐BB hydrolysis yielded exclusively the monomeric reaction product and occurred with a 25‐fold higher catalytic efficiency than BB single hydrolysis. Thus, ferri‐BB was the preferred substrate of the YuiI esterase whose gene locus was designated besA.

Inhibition of aryl acid adenylation domains involved in bacterial siderophore synthesis

Aryl acid adenylation domains are the initial enzymes for aryl‐capping of catecholic siderophores in a plethora of microorganisms. In order to overcome the problem of iron acquisition in host organisms, siderophore biosynthesis is decisive for virulence development in numerous important human and animal pathogens. Recently, it was shown that growth of Mycobacterium tuberculosis and Yersinia pestis can be inhibited in an iron‐dependent manner using the arylic acyl adenylate analogue 5′‐O‐[N‐(salicyl)‐sulfamoyl] adenosine that acts on the salicylate activating domains, MbtA and YbtE [Ferreras JA, Ryu JS, Di Lello F, Tan DS, Quadri LEN (2005) Nat Chem Biol1, 29–32]. The present study explores the behaviour of the 2,3‐dihydroxybenzoate activating domain DhbE (bacillibactin synthesis) and compares it to that of YbtE (yersiniabactin synthesis) upon enzymatic inhibition using a set of newly synthesized aryl sulfamoyl adenosine derivatives. The obtained results underline the highly specific mode of inhibition for both aryl acid activating domains in accordance with their natively accepted aryl moiety. These findings are discussed regarding the structure–function based aspect of aryl substrate binding to the DhbE and YbtE active sites.

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

We identified a Cu accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulated Cu, dependent on the nutritional Cu sensor CRR1, but was functionally Cu-deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. NanoSIMS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy (XAS) was consistent with Cu+ accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu+ became bio-available for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mis-metallation during Zn deficiency and enabling efficient cuproprotein (re)-metallation upon Zn resupply.

SufU Is an Essential Iron-Sulfur Cluster Scaffold Protein in Bacillus subtilis

Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF, and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In Escherichia coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in Bacillus subtilis suggests that this bacteriums genome encodes only a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted of SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total-cell lysates, suggesting a crucial function of SufU in Fe/S protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to approximately 1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS-dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold protein tightly cooperating with the SufS cysteine desulfurase.

Involvement of Bacillus subtilis ClpE in CtsR degradation and protein quality control

The heat-inducible CtsR regulon of Bacillus subtilis codes for three Clp proteins with chaperone or protease activity. While the importance of ClpC and ClpP has been elucidated for a wide range of cellular adaptation processes, this study deals with the physiological role of B. subtilis ClpE. Northern experiments and reporter gene analyses revealed that ClpE is essential both for efficient CtsR-dependent gene derepression and for rerepression during heat stress. ClpEP was found to destabilize the global regulator CtsR after heat shock in vivo with different kinetics than ClpCP, which is known to degrade CtsR in vitro and in vivo upon heat stress. Furthermore, ClpE was localized at heat-generated inclusion bodies by electron microscopy. The comparison of radiolabeled aggregated protein fractions of wild-type and clpE mutant cells during heat stress displayed a significant delay of protein disaggregation in the absence of ClpE. A kinetic Western blotting approach confirmed the long-term residence of ClpE in the insoluble cell fraction rather than in the cytoplasmic fraction. These observations indicate the involvement of ClpE in global protein disaggregation. As a characteristic structural element of ClpE, the N-terminal zinc finger domain was proven to be essential for basal in vitro ATPase activity.

Copper acquisition is mediated by YcnJ and regulated by YcnK and CsoR in Bacillus subtilis.

Copper is an essential cofactor for many enzymes, and at over a threshold level, it is toxic for all organisms. To understand the mechanisms underlying copper homeostasis of the gram-positive bacterium Bacillus subtilis, we have performed microarray studies under copper-limiting conditions. These studies revealed that the ycnJ gene encodes a protein that plays an important role in copper metabolism, as it shows a significant, eightfold upregulation under copper-limiting conditions and its disruption causes a growth-defective phenotype under copper deprivation as well as a reduced intracellular content of copper. Native gel shift experiments with the periplasmic N-terminal domain of the YcnJ membrane protein (135 residues) disclosed its strong affinity to Cu(II) ions in vitro. Inspection of the upstream sequence of ycnJ revealed that the ycnK gene encodes a putative transcriptional regulator, whose deletion caused an elevated expression of ycnJ, especially under conditions of copper excess. Further studies demonstrated that the recently identified copper efflux regulator CsoR also is involved in the regulation of ycnJ expression, leading to a new model for copper homeostasis in B. subtilis.

The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron

Efficient uptake of iron is of critical importance for growth and viability of microbial cells. Nevertheless, several mechanisms for iron uptake are not yet clearly defined. Here we report that the widely conserved transporter EfeUOB employs an unprecedented dual-mode mechanism for acquisition of ferrous (Fe[II]) and ferric (Fe[III]) iron in the bacterium Bacillus subtilis. We show that the binding protein EfeO and the permease EfeU form a minimal complex for ferric iron uptake. The third component EfeB is a hemoprotein that oxidizes ferrous iron to ferric iron for uptake by EfeUO. Accordingly, EfeB promotes growth under microaerobic conditions where ferrous iron is more abundant. Notably, EfeB also fulfills a vital role in cell envelope stress protection by eliminating reactive oxygen species that accumulate in the presence of ferrous iron. In conclusion, the EfeUOB system contributes to the high-affinity uptake of iron that is available in two different oxidation states.

Structural Basis and Stereochemistry of Triscatecholate Siderophore Binding by Feua.

The acquisition of iron is a key feature of microbial growth, in particular for the development of virulence in animal and human hosts. The secretion of low-molecular-weight organic chelators called siderophores is one of the main ironmobilizing strategies. The strict homeostasis of iron in mammalians led in particular to the development of chelators with extremely high affinity. 2] The two strongest chelators are represented by the triscatecholate-trilactone derivatives bacillibactin and enterobactin (H6-BB and H6-Ent, respectively; Scheme 1), which have formation constants of 10 and 10m , respectively. 4] As a result of their great importance in pathogenicity, both siderophores are targets of the innate defense system that acts by siderocalin (NGAL, lipocalin 2) dependent sequestration. 6] Bacillus anthracis, B. cereus, and nonpathogenic relatives such as B. subtilis secrete bacillibactin, a cyclic trilactone depsipeptide, comprising three subunits of the 2,3-dihydroxybenzoate (2,3-DHB)-Gly-Thr, which are assembled by a nonribosomal peptide synthetase (NRPS). Cellular uptake of ferribacillibactin ([Fe(BB)] ) relies on the ATP-binding cassette transporter FeuABC-YusV, which is also able to import [Fe(Ent)] . 9] A comprehension of the coevolution of hosts and pathogens in regard to siderophore scavenging requires a precise understanding of existing siderophore–protein interactions. These may help to create novel strategies for defense against pathogens, for example, by siderophore–drug design or by affinity engineering of available binding pockets. We report here the 1.7 crystal structure of the siderophore binding protein FeuA from B. subtilis in a complex with [Fe(BB)] . Detailed analysis of the protein–ligand interactions at high structural resolution is complemented by fluorescence and CD spectroscopic studies on variants of the binding site and ligand configuration. The siderophore binding protein FeuA (297 amino acids without a signal peptide) is attached to the cytoplasmic membrane by a lipid anchor tethered to the N-terminal cysteine residue of the mature protein. We obtained crystals in different space groups of FeuA without the lipid anchor (lacking the first 20 amino acids) with and without [Fe(BB)] , and solved the phases by molecular replacement (see the Supporting Information). Basically, FeuA is composed of two domains, which show a Rossmann-like fold and are connected by a 22 amino acid long a helix (Figure 1). These structural elements are indicative of siderophore binding proteins of the “helical-backbone” metal-receptor superfamily such as FhuD and CeuE. 11] The binding of the substrate at the interface of the Nand C-terminal domains induces a movement towards the binding site. Superposition of the N-terminal domains of apoand holo-FeuA results in a shift of 20.28 in the C-terminal domain (Figure 2). This bending is not as large as that observed for binding proteins with flexible antiparallel b-strand linkers, such as the maltose binding protein, but larger than in other Scheme 1. Bacillibactin, its relative enterobactin, and the catecholate siderophore mimic mecam.