Produção Nacional
Revisado por pares
Fluorescent sensors of siderophores produced by bacterial pathogens
2022; Elsevier BV; Volume: 298; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2022.101651
ISSN1083-351X
AutoresAshish Kumar, Taihao Yang, Somnath Chakravorty, Aritri Majumdar, Brittany L. Nairn, David A. Six, Naara M. dos Santos, Sarah L. Price, Matthew B. Lawrenz, Luis A. Actis, Marilis V. Marques, Thomas A. Russo, Salete M. Newton, Phillip E. Klebba,
Tópico(s)Plant Pathogenic Bacteria Studies
ResumoSiderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria. Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria. 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Toward those ends, the production of a fluorescent LGP (FLGP) in a transport-deficient, ΔtonB host creates a “decoy” sensor cell that detects and quantifies ligands in experimental suspensions or solutions. We used this strategy to produce 18 FLGP sensors and two fluorescent binding protein (FBP) sensors from siderocalin and EcoFepB. Collectively, they detected siderophore biosynthesis, measured siderophore concentrations, and monitored ferric siderophore uptake by cells of interest. We engineered FLGP sensors of ferric catecholates (Ent, GEnt, dihydroxybenzoate [DHB], dihydroxybenzoyl serine [DHBS], cefidericol [FDC; (55Ito A. Nishikawa T. Matsumoto S. Yoshizawa H. Sato T. Nakamura R. Tsuji M. Yamano Y. Siderophore cephalosporin cefiderocol utilizes ferric iron transporter systems for antibacterial activity against Pseudomonas aeruginosa.Antimicrob. Agents Chemother. 2016; 60: 7396-7401Google Scholar, 56Kohira N. West J. Ito A. Ito-Horiyama T. Nakamura R. Sato T. Rittenhouse S. 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We made additional FBP sensors from EcoFepB, a periplasmic binding protein that acts in ferric catecholate uptake, and human siderocalin (HsaSCN), which recognizes multiple siderophores. We cloned the LGP structural genes in the low-copy plasmid pHSG575 (63Hashimoto-Gotoh T. Franklin F.C. Nordheim A. Timmis K.N. Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors.Gene. 1981; 16: 227-235Google Scholar, 64Scott D.C. Cao Z. Qi Z. Bauler M. Igo J.D. Newton S.M. Klebba P.E. Exchangeability of N termini in the ligand-gated porins of Escherichia coli.J. Biol. Chem. 2001; 276: 13025-13033Google Scholar, 65Takeshita S. Sato M. Toba M. Masahashi W. Hashimoto-Gotoh T. High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection.Gene. 1987; 61: 63-74Google Scholar) and the binding protein genes in pET28a-c (+), that added a 6-His tag to their N termini. After verifying the clones by DNA sequencing, we changed a handful of residues in each protein to cysteine. Nearly all of these OM proteins are devoid of cysteine; the few exceptions contain Cys pairs that are unreactive unless subject to reduction by β-mercaptoethanol or other chemical agents (66Liu J. Rutz J.M. Klebba P.E. Feix J.B. A site-directed spin-labeling study of ligand-induced conformational change in the ferric enterobactin receptor, FepA.Biochemistry. 1994; 33: 13274-13283Google Scholar). We chose the target residues for site-directed Cys mutagenesis from analysis of crystal structures (EcoFepA, EcoCir, EcoFiu, EcoFhuA, EcoBtuB, KpnFyuA, AbaPiuA, AbaPirA, AbaBauA, HsaSCN) or by their location in hypothetical structures predicted by the Modeller algorithm of CHIMERA (CcrHutA, EcoIutA, KpnFepA, KpnIroN, AbaFepA, PaeFepA). After construction and verification, we expressed the proteins in E. coli, subjected each Cys mutant LGP to modification by fluorescein maleimide (FM; 5 μM for 15 min at 37 °C; (67Smallwood C.R. Jordan L. Trinh V. Schuerch D.W. Gala A. Hanson M. Shipelskiy Y. Majumdar A. Newton S.M. Klebba P.E. Concerted loop motion triggers induced fit of FepA to ferric enterobactin.J. Gen. Physiol. 2014; 144: 71-80Google Scholar)), and then analyzed their expression, fluoresceination, and fluorescence quenching during binding of a metal complex. Relative to pathogenic host organisms, the production of FLGP in nonpathogenic, rough (rfaB) E. coli K-12 facilitated their optimum FM labeling in a cell surface environment that is unobscured by LPS O-antigen or capsule. We obtained high-level, usually stoichiometric modification of the engineered Cys sulfhydryls with extrinsic maleimide fluorophores (Figs. S2–S6). Certain Cys locations were better or worse for some LGP (e.g., AbaBauA, EcoFiu), but we always found accessible labeling sites in each OM protein. We then chose the most sensitive single mutant of each LGP and spectroscopically determined its specificities and affinities for the apo- and ferric siderophores in relevant chemical classes (Table 1). For production of HsaSCN and EcoFepB, we introduced Cys substitutions at positions near their binding sites, overexpressed the proteins, and purified them from cell lysates by metal ion chromatography (Fig. S6). For each LGP or soluble binding protein sensor, we studied three or more Cys substitutions to identify an effective site for attachment of an extrinsic fluorophore. To optimize detection and quantification of the individual metal complexes, we compared the fluorescence intensities of the different Cys mutant derivatives, as well as the extents of their quenching by their homologous and heterologous ligands.Table 1Affinities (KD values; nM or uM) of LGP sensors for apo and ferric siderophores and porphyrins1LGP acronyms abbreviate the genus and species of their origin: E. coli FepA, EcoFepA; K. pneumoniae FepA locus 1658, KpnFepA1658; Yersinia pestis FyuA, YpeFyuA; Homo sapien SCN, HsaSCN, etc.2Location of the Cys substitution in the mature protein sequence.3The table lists KD values (blue text: nM; black text: uM) from analysis of fluorescence quenching with ferric or apo (parenthetic values) siderophores or porphyrins (see Figure 1, Figure 2, Figure 3). We performed each quenching titration 2 or 3 times; the tabulated KD values result from a single representative experiment and are listed with their associated standard errors from a nonlinear curve fit to a single-site binding model, using Grafit 6.012 (Erithacus Ltd, Middlesex, UK). The KD values of preferred natural ligands are underlined. FeEnt∗, partially degraded FeEnt; NB, no binding; ND, no data. Open table in a new tab 1LGP acronyms abbreviate the genus and species of their origin: E. coli FepA, EcoFepA; K. pneumoniae FepA locus 1658, KpnFepA1658; Yersinia pestis FyuA, YpeFyuA; Homo sapien SCN, HsaSCN, etc. 2Location of the Cys substitution in the mature protein sequence. 3The table lists KD values (blue text: nM; black text: uM) from analysis of fluorescence quenching with ferric or apo (parenthetic values) siderophores or porphyrins (see Figure 1, Figure 2, Figure 3). We performed each quenching titration 2 or 3 times; the tabulated KD values result from a single representative experiment and are listed with their associated standard errors from a nonlinear curve fit to a single-site binding model, using Grafit 6.012 (Erithacus Ltd, Middlesex, UK). The KD values of preferred natural ligands are underlined. FeEnt∗, partially degraded FeEnt; NB, no binding; ND, no data. The high (nanomolar) affinity of certain LGPs for a particular ligand suggested potential exclusivity in their binding reactions. EcoFepA, for example, tightly adsorbs FeEnt (KD = 0.1–0.4 nM (53Chakravorty S. Shipelskiy Y. Kumar A. Majumdar A. Yang T. Nairn B.L. Newton S.M. Klebba P.E. Universal fluorescent sensors of high-affinity iron transport, applied to ESKAPE pathogens.J. Biol. Chem. 2019; 294: 4682-4692Google Scholar, 68Cao Z. Qi Z. Sprencel C. Newton S.M. Klebba P.E. Aromatic components of two ferric enterobactin binding sites in Escherichia coli fepA.Mol. Microbiol. 2000; 37: 1306-1317Google Scholar, 69Cao Z. Warfel P. Newton S.M. Klebba P.E. Spectroscopic observations of ferric enterobactin transport.J. Biol. Chem. 2003; 278: 1022-1028Google Scholar, 70Newton S.M. Igo J.D. Scott D.C. Klebba P.E. Effect of loop deletions on the binding and transport of ferric enterobactin by FepA.Mol. Microbiol. 1999; 32: 1153-1165Google Scholar)), which allows it to scavenge subnanomolar concentrations of FeEnt. However, we found t
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