Fluorescent sensors of siderophores produced by bacterial pathogens.

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.

Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium.

In response to iron deprivation, Salmonella enterica serovar Typhimurium secretes two catecholate-type siderophores, enterobactin and its glucosylated derivative salmochelin. Although the systems responsible for enterobactin synthesis and acquisition are well characterized, the mechanisms of salmochelin secretion and acquisition, as well as its role in Salmonella virulence, are incompletely understood. Herein we show by liquid chromatography-mass spectrometry analysis of culture supernatants from wild type and isogenic mutant bacterial strains that the Major Facilitator Superfamily pump EntS is the major exporter of enterobactin and the ABC transporter IroC exports both salmochelin and enterobactin. Growth promotion experiments demonstrate that IroC is not required for utilization of Fe-enterobactin or Fe-salmochelin, as had been previously suggested, but the ABC transporter protein FepD is required for utilization of both siderophores. Salmonella mutants deficient in salmochelin synthesis or secretion exhibit reduced virulence during systemic infection of mice.

The detection of salmochelin and yersiniabactin in uropathogenic Escherichia coli strains by a novel hydrolysis-fluorescence-detection (HFD) method.

Escherichia coli strains produce a variety of structurally different siderophores of which enterobactin, aerobactin and yersiniabactin have been reported earlier to occur in strains of extraintestinal infections. In uropathogenic E. coli (UPEC) strains novel siderophores, named salmochelins, have recently been identified which contain C-glucosylated 2,3-dihydroxybenzoyl-L-serine (glucosyl-DHB-serine) residues connected in a linear (mono-, di- , trimeric) or cyclic form. We report here on a fast and simple hydrolysis-fluorescence-detection (HFD) method, based on identification of C-glucosylated dihydroxybenzoic acid (glucosyl-DHB). Salmochelin containing culture filtrates were bound to DEAE cellulose spin columns, hydrolyzed and the breakdown products were subsequently identified by HPLC or thin layer chromatography (TLC). The hydrolysis products can be easily detected by their fluorescence, either during HPLC separation connected to a fluorescence detector or after TLC on cellulose plates viewed under a UV254 or UV365 lamp. While DHB originates from the hydrolysis of enterobactin and salmochelin, glucosyl-DHB is only found as a characteristic hydrolysis product of salmochelins (S1, S2, S4). The HFD method allows detection of salmochelin in the presence of other siderophores, such as enterobactin, aerobactin and yersiniabactin. Several clinical UPEC isolates containing the iroN gene cluster were analyzed by this procedure, showing that all isolates were glucosyl-DHB positive indicating salmochelin production, while a collection of other pathogenic E. coli strains (EHEC, EIEC, ETEC, EAggEC and EPEC) were glucosyl-DHB negative. In addition, the HFD method allowed the identification of yersiniabactin due to a fluorescent salicylate-containing degradation product.

Enterobactin: the characteristic catecholate siderophore of Enterobacteriaceae is produced by Streptomyces species.(1).

Enterobactin is described in the literature as the typical iron-chelating compound (siderophore) produced by bacteria of the family Enterobacteriaceae. In the course of a HPLC with diode array detection screening programme for detection of novel secondary metabolites, enterobactin, its biosynthetic precursor 2,3-dihydroxy-N-benzoylserine and its linear dimer and trimer condensation products were found to be produced by two Streptomyces strains besides the trihydroxamate-type siderophores desferri-ferrioxamine B and E.

Potential virulence determinants of Salmonella serovars from poultry and human sources in Spain.

A total of 173 Salmonella strains of different serovars isolated during 1992 from poultry and human sources in Zaragoza (NE Spain) were investigated for potential virulence factors. Parameters studied included production of aerobactin, enterobactin, colicin (including colicin V) and hemolysin, serum resistance against serum from man, sheep, cattle and chicken, binding of Congo red and crystal violet, auto-agglutination and calcium dependency at 37 degrees C and double colony morphology. Preliminary studies showed tests with completely negative results (colicins and hemolysin production, double colony morphology, auto-agglutination and calcium dependency at 37 degrees C) and tests with completely positive results (enterobactin production and binding of Congo red). The tests with variability of results were production of aerobactin, serum resistance and crystal violet binding. Aerobactin production was detected in 80% of Salmonella strains of clinical human origin and in 30% of Salmonella strains isolated from healthy slaughtered chickens. Sixty-five per cent of patient isolates were human serum resistant, 73% were ovine serum resistant, 85% were bovine serum resistant and 98% were chicken serum resistant. The percentages of poultry isolates serum resistant were 61, 86, 60 and 89% in human, ovine, bovine and chicken serum, respectively. Crystal violet binding was detected in 22.5% of Salmonella isolates from human origin and in 32.3% of Salmonella strains from poultry origin. This study can form a marker for the prevalence of strains with various characteristics (production of aerobactin, serum resistance and crystal violet binding) for comparison in future epidemiological studies. Furthermore, the data of this work suggested that strains causing enteric salmonellosis in man are partially identical to strains isolated from carrier broilers.

Iron supplying systems of Salmonella in diagnostics, epidemiology and infection.

Well-known and newly characterised mechanisms, both endogenous and exogenous, for the uptake of iron by Salmonella are outlined, and their possible roles at various stages in infection are discussed. The contributions of a detailed understanding of iron supplying systems to techniques for diagnosis, epidemiology and disease management are described.

Serotypes, hemagglutinins, siderophore synthesis, and serum resistance of Klebsiella isolates causing human urinary tract infections.

A total of 146 Klebsiella isolates from human asymptomatic bacteriuria (n = 73), cystitis (n = 54), and acute pyelonephritis (n = 19) were examined for the presence of particular virulence factors. Capsular type K2 was the most common serotype observed (13%). This capsule type was prevalent in isolates from asymptomatic bacteriuria and cystitis but not from pyelonephritis. Type 1 fimbriae were found significantly more often in pyelonephritis isolates than among those from asymptomatic and symptomatic lower urinary tract infection (UTI; P < .05), while no marked differences were detected with respect to the distribution of type 3 fimbriae. Serum resistance was more frequent among isolates from symptomatic (26%) than from asymptomatic UTI (18%). Enterochelin was produced by all but 1 of the isolates as determined by a bioassay. In contrast, aerobactin synthesis was rare (3%), with isolates from pyelonephritis showing the highest frequency of aerobactin production (3/19).

Role of aerobactin in systemic spread of an opportunistic strain of Escherichia coli from the intestinal tract of gnotobiotic lambs.

To assess the role of the aerobactin-related system in the virulence of bovine opportunistic Escherichia coli, and to determine the stage(s) of the overall infectious process at which it is acting, germfree lambs were mixedly infected orally with two derivative strains of this bacterium differing in their ability (Iut+) or inability (Iut-) to express a functional aerobactin-mediated iron transport system. The Iut- strain was compared with the Iut+ strain for colonization of the gut, translocation to the mesenteric lymph nodes (MLN), and spread to other organs and to the body fluids of diassociated lambs. The Iut- mutant was found in smaller numbers in the duodenum, suggesting that aerobactin conferred a significant selective advantage for colonization of this intestinal segment. Although the two challenge strains translocated to MLN, the population level in the MLN was always higher for the Iut+ strain. Moreover, experimental infections resulted in recovery of only the Iut+ strain in the organs other than the MLN and in the body fluids. These results indicate a role for aerobactin in promoting systemic spread of the bacteria from the intestine. Direct evidence was obtained that aerobactin secretion occurred in vivo at both intestinal and extraintestinal sites of infection. In contrast to enterobactin, aerobactin was detected in the duodenum, jejunum, ileum, cecum, liver, spleen, kidney, urine, cerebrospinal fluid, and bile. The highest concentration of aerobactin was found in the urine, even when the samples were devoid of infecting bacteria. All of these findings suggest that aerobactin is released in vivo in a diffusible form and that it may be an important step in the production of disease by intestinal opportunistic E. coli.

[A study of some pathogenic factors of Salmonella typhi]. / Estudio de algunos factores de patogenicidad de la Salmonella typhi.

30 strains isolated from clinical cases and a laboratory induced R-mutant or S typhi were studied. The synthesis of Colicin V, Siderophores, hydroxamate and phenolate (Aerobactin and Enterochelin) were investigated using biological assays. All strains were positive for Enterochelin but only strains 6586 and 4448 were positive for Aerobactin. An 80 kD receptor for enterochelin was found in the outer membrane of strains. Only strains 4448, 635 and 4693 produced Colicin V. Plasmid related antibiotic resistance was demonstrated in 7 strains and was considered "cryptic" in 3 others. The LD50 for mice ranged from 2.7 x 10(4) to 1 x 10(9) bacteria per ml. The presence of pathogenic factors was not related to the LD50.

Different systems for iron supply of Salmonella typhimurium and Escherichia coli wild strains as tool for typing.

Hitherto the siderophore-pattern analysis included bioassays to detect enterobactin,aerobactin and other siderophores of Enterobacteriaceae. In addition, 2,3-dihydroxybenzoic acid, the precursor of enterobactin, and new hydroxamate siderophores could be examined by means of two new Salmonella mutants as indicator strains. In this manner the siderophore pattern analysis extends its discriminating ability. Among 167 Salmonella typhimurium strains tested, we detected 5 siderophore patterns. 6 siderophore patterns could be detected among a total of 204 E. coli strains. Using siderophore pattern analysis for clinical-epidemiological and ecological purposes appropriate technical methods can be recommended.

Identification of enterobactin and linear dihydroxybenzoylserine compounds by HPLC and ion spray mass spectrometry (LC/MS and MS/MS).

The production of catecholate siderophores was studied in some selected species of Enterobacter (Enterobacteriaceae). The extracted catecholates were separated as iron-free compounds by HPLC on a C18 reversed-phase column using methanol/0.1% phosphoric acid or methanol/0.1% formic acid as a solvent system and identified by ion spray mass spectrometry (LC/MS, MS/MS). Five catecholate compounds were identified which include 2,3-dihydroxybenzoylserine, its linear dimer and trimer, the cyclic enterobactin and an unidentified isomer of enterobactin. In addition, a new large-scale method for the isolation of catecholate siderphores is described which is based on adsorption on XAD-2 and subsequent purification on Sephadex LH20.

The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product.

The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a transmembrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA-entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5' to entD, along with RNA hybridization data, suggest that an iron-regulated transcript contains both fepA and entD.

Nucleotide sequence of the Escherichia coli entE gene.

The Escherichia coli entE gene encodes a polypeptide necessary in the latter stages of biosynthesis of the siderophore enterobactin. The entE gene and adjacent DNA were sequenced. The predicted EntE polypeptide consists of 536 amino acids and has a Mr of 58,299 and a net charge of -7.33. Genetic evidence combined with this and previous sequencing data indicate that the genes entCEB(G)A are transcribed as unit from a promoter upstream of entC.

Bactericidal activity of M14659 enhanced in low-iron environments.

The bactericidal activity of M14659 against Escherichia coli in low-iron environments was investigated and compared with that of ceftriaxone and ceftazidime. The bactericidal activity of M14659 against E. coli in Mueller-Hinton broth was enhanced 30- to 20,000-fold by addition of transferrin, which is an iron-binding protein, whereas the activity of ceftriaxone or ceftazidime was much less strongly affected. This enhancement by transferrin was completely inhibited by saturating the iron-binding capacity of transferrin with FeCl3. M14659 was taken up markedly into bacterial cells in the presence of transferrin, and its uptake was inhibited by the protonophore dinitrophenol, which inhibits active-transport systems coupled to an energized membrane such as the iron transport systems of E. coli. The bactericidal activity of M14659, which chelates Fe3+, was also enhanced in the presence of other iron-binding compounds such as lactoferrin and alpha,alpha'-dipyridyl or in iron-deficient Mueller-Hinton broth (Fe3+ concentration, less than 2 nM) supplemented with FeCl3 at 0.1 to 1.0 microM, but not in unsupplemented iron-deficient Mueller-Hinton broth. The E. coli used in this study was confirmed to derepress iron transport systems in the presence of transferrin, lactoferrin, and alpha,alpha'-dipyridyl and in the iron-deficient Mueller-Hinton broth supplemented with FeCl3 at 0 to 1.0 microM. M14659 also showed an excellent antibacterial activity in vitro against other gram-negative bacteria in the low-iron environments. These findings indicate that M14659 may be actively taken up with Fe3+ into bacterial cells, probably through the iron transport systems under conditions of low iron and, thus, kills bacteria effectively.

Further differentiation of Enterobacteriaceae by means of siderophore-pattern analysis.

By means of a combination of 5 siderophore bioassays using several indicator strains, genera, species and subspecies of Enterobacteriaceae can be further differentiated. Enterobactin, aerobactin and other siderophores produced can be detected. Each strain shows specific pattern which we called siderophore-pattern. It is easy to separate Morganella, Proteus, Providencia, Yersinia strains from the genera Salmonella, Shigella, Escherichia coli, Enterobacter, Citrobacter, Klebsiella, Serratia. Enterobacter agglomerans strains differ from other Enterobacter species with respect to their siderophore pattern. In Salmonella strains there are differences between the subspecies I and IV. Additionally the most strains of Salmonella subspecies I from nosocomial infections produced aerobactin, in the most cases determined by plasmids. Among Shigella strains different siderophore pattern exist according to other epidemiological markers. S. flexneri strains of serovar 6 produced contrary to the strains of other serovars enterobactin. By means of the siderophore-pattern analysis E. coli strains of serovars 01; 02; 018 can be further differentiated. E. coli 01:K1 strains containing the fimbrial antigen F11 produced aerobactin whereas the F9 strains did not. All Hafnia alvei strains produced a uniform siderophore pattern, different from all other members of the enterobacteriaceae family. With this aim Hafnia alvei strains can be easily separated under practical conditions.

Novel iron uptake system specified by ColV plasmids: an important component in the virulence of invasive strains of Escherichia coli.

The enhanced virulence of invasive strains of Escherichia coli carrying ColV plasmids was shown to be due to a novel plasmid-mediated iron uptake system. Possession of a ColV plasmid conferred strong selective advantage on the host bacterial strain in experimental infections unless excess iron was administered in the inoculum. Moreover, supplementation of defined minimal medium with transferrin to complex available iron caused marked limitation of the growth of plasmid-free strains but had no effect on strains carrying a ColV plasmid. The activity of an efficient iron uptake process was clearly shown by experiments with a mutant of E. coli deficient in enterochelin biosynthesis. Although the mutant was dependent on the presence of citrate in the growth medium to facilitate iron transport, colicinogenic derivatives did not require added citrate for growth. Radioactive iron was shown to be taken up rapidly by nongrowing cells of the plasmid-carrying strain. Furthermore, it was observed that repression of the synthesis of specific outer membrane proteins normally induced by conditions of iron deficit was maintained after a shift of the colicinogenic strains from a rich medium to a medium low in iron. The ColV plasmid-mediated iron uptake system was independent of the active iron transport mechanisms known in E. coli, but like them it required tonB activity as a source of energy.