A quorum-sensing regulatory cascade for siderophore-mediated iron homeostasis in Chromobacterium violaceum.

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

Ferrous iron uptake via IRT1/ZIP evolved at least twice in green plants.

Iron (Fe) is essential for virtually all organisms, being irreplaceable because of its electrochemical properties that enable many biochemical processes, including photosynthesis. Besides its abundance, Fe is generally found in the poorly soluble form of ferric iron (Fe3+ ), while most plants uptake the soluble form Fe2+ . The model angiosperm Arabidopsis thaliana, for example, captures Fe through a mechanism that lowers rhizosphere pH through proton pumping that increases Fe3+ solubility, which is then reduced by a membrane-bound reductase and transported into the cell by the zinc-regulated, iron-regulated transporter-like protein (ZIP) family protein AtIRT1. ZIP proteins are transmembrane transporters of divalent metals such as Fe2+ , Zn2+ , Mn2+ , and Cd2+ . In this work, we investigated the evolution of functional homologs of IRON-REGULATED TRANSPORTER 1/ZIP in the supergroup Archaeplastida (Viridiplantae + Rhodophyta + Glaucophyta) using 51 genomes of diverse lineages. Our analyses suggest that Fe is acquired through deeply divergent ZIP proteins in land plants and chlorophyte green algae, indicating that Fe2+ uptake by ZIP proteins evolved independently at least twice throughout green plant evolution. Our results indicate that the archetypical IRON-REGULATED TRANSPORTER (IRT) proteins from angiosperms likely emerged before the origin of land plants during early streptophyte algae terrestrialization, a process that required the evolution of Fe acquisition in terrestrial subaerial settings.

MliR, a novel MerR-like regulator of iron homeostasis, impacts metabolism, membrane remodeling, and cell adhesion in the marine Bacteroidetes Bizionia argentinensis.

The MerR family is a group of transcriptional activators with conserved N-terminal helix-turn-helix DNA binding domains and variable C-terminal effector binding regions. In most MerR proteins the effector binding domain (EBD) contains a cysteine center suited for metal binding and mediates the response to environmental stimuli, such as oxidative stress, heavy metals or antibiotics. We here present a novel transcriptional regulator classified in the MerR superfamily that lacks an EBD domain and has neither conserved metal binding sites nor cysteine residues. This regulator from the psychrotolerant bacteria Bizionia argentinensis JUB59 is involved in iron homeostasis and was named MliR (MerR-like iron responsive Regulator). In silico analysis revealed that homologs of the MliR protein are widely distributed among different bacterial species. Deletion of the mliR gene led to decreased cell growth, increased cell adhesion and filamentation. Genome-wide transcriptomic analysis showed that genes associated with iron homeostasis were downregulated in mliR-deletion mutant. Through nuclear magnetic resonance-based metabolomics, ICP-MS, fluorescence microscopy and biochemical analysis we evaluated metabolic and phenotypic changes associated with mliR deletion. This work provides the first evidence of a MerR-family regulator involved in iron homeostasis and contributes to expanding our current knowledge on relevant metabolic pathways and cell remodeling mechanisms underlying in the adaptive response to iron availability in bacteria.

Detection of Fur, AmoA and pvcAB genes in Aeromonas hydrophila isolated from aquatic organisms and impact on bacterial growth under different iron concentrations / Detecção dos genes Fur, AmoA e pvcAB em Aeromonas hydrophila isoladas de organismos aquáticos e o impacto da presença desses genes sobre o crescimento bacteriano com diferentes concentrações de ferro

Infection caused by Aeromonas brings great harm to fish farming. Among the factors associated with bacterial pathogenesis, iron uptake can contribute to the survival and virulence of bacteria within hosts. The aim of this study was to check the presence of genes related to iron uptake in Aeromonas hydrophila deriving from aquatic organisms in the São Francisco Valley and associate the presence of these genes with the ability to grow in media containing different concentrations of iron. The DNAs of 41 isolates were extracted and used in PCRs to verify the presence of the Fur, AmoA and pvcAB genes related to iron uptake. The growth of the isolates belonging to different genetic profiles was verified in culture media containing different iron concentrations. Two isolates were positive for the presence of the Fur gene, seven for the AmoA gene and two for the pvcAB gene. The growth test showed that the low availability of iron did not interfere in the growth of the isolates, nor in the isolate that did not contain any of the genes evaluated in this study, suggesting that the iron uptake's mechanisms of the tested isolates may be related to other genes and proteins.

Infecções causadas por Aeromonas trazem grandes prejuízos à piscicultura. Entre os fatores associados à patogênese bacteriana, a captação de ferro pode contribuir para a sobrevivência e a virulência das bactérias dentro dos hospedeiros. O objetivo deste estudo foi verificar a presença de genes relacionados à captação de ferro em Aeromonas hydrophila provenientes de organismos aquáticos do Vale do São Francisco e associar a presença desses genes com a capacidade de as bactérias crescerem em meios contendo diferentes concentrações de ferro. Os DNAs de 41 isolados foram extraídos e utilizados em PCRs para verificar a presença dos genes Fur, AmoA e pvcAB relacionados à captação de ferro. O crescimento dos isolados pertencentes a diferentes perfis genéticos foi verificado em meios de cultura contendo diferentes concentrações de ferro. Dois isolados foram positivos para a presença do gene Fur, sete para a do gene AmoA e dois para a do gene pvcAB. O teste de crescimento mostrou que a baixa disponibilidade de ferro não interferiu no crescimento dos isolados nem no isolado que não continha nenhum dos genes avaliados neste estudo, sugerindo que os mecanismos de captação de ferro dos isolados testados podem estar relacionados a outros genes e proteínas.

The "Little Iron Waltz": The Ternary Response of Paracoccidioides spp. to Iron Deprivation.

Paracoccidioides is a genus of thermodimorphic fungi that causes paracoccidioidomycosis. When in the host, the fungus undergoes several challenges, including iron deprivation imposed by nutritional immunity. In response to the iron deprivation triggered by the host, the fungus responds in a ternary manner using mechanisms of high affinity and specificity for the uptake of Fe, namely non-classical reductive iron uptake pathway, uptake of host iron proteins, and biosynthesis and uptake of siderophores. This triple response resembles the rhythmic structure of a waltz, which features three beats per compass. Using this connotation, we have constructed this review summarizing relevant findings in this area of study and pointing out new discoveries and perspectives that may contribute to the expansion of this "little iron waltz".

Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea.

The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.

Production and Uptake of Distinct Endogenous Catecholate-Type Siderophores Are Required for Iron Acquisition and Virulence in Chromobacterium violaceum.

Bacteria use siderophores to scavenge iron from environmental or host sources. The iron acquisition systems of Chromobacterium violaceum, a ubiquitous environmental bacterium that can cause infections in humans, are still unknown. In this work, we demonstrated that C. violaceum produces putative distinct endogenous siderophores, here named chromobactin and viobactin, and showed that they are each required for iron uptake and virulence. An in silico analysis in the genome of C. violaceum revealed that genes related to synthesis and uptake of chromobactin (cba) and viobactin (vba) are located within two secondary-metabolite biosynthetic gene clusters. Using a combination of gene deletions and siderophore detection assays, we revealed that chromobactin and viobactin are catecholate siderophores synthesized from the common precursor 2,3-dihydroxybenzoate (2,3-DHB) on two nonribosomal peptide synthetase (NRPS) enzymes (CbaF and VbaF) and taken up by two TonB-dependent receptors (CbuA and VbuA). Infection assays in mice revealed that both the synthesis and the uptake of chromobactin or viobactin are required for the virulence of C. violaceum, since only the mutant strains that do not produce any siderophores or are unable to take up both of them were attenuated for virulence. In addition, the mutant strain unable to take up both siderophores showed a pronounced attenuation of virulence in vivo and reduced neutrophil extracellular trap (NET) formation in in vitro assays, suggesting that extracellularly accumulated siderophores modulate the host immune response. Overall, our results revealed that C. violaceum uses distinct endogenous siderophores for iron uptake and its establishment in the host.

The Fish Pathogen Vibrio ordalii Under Iron Deprivation Produces the Siderophore Piscibactin.

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using ß-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

Herbaspirillum seropedicae Differentially Expressed Genes in Response to Iron Availability.

Herbaspirillum seropedicae Z67 is a nitrogen-fixing endophyte that colonizes many important crops. Like in almost all organisms, vital cellular processes of this endophyte are iron dependent. In order to efficiently acquire iron to fulfill its requirements, this bacterium produces the siderophores serobactins. However, the presence in its genome of many others iron acquisition genes suggests that serobactins are not the only strategy used by H. seropedicae to overcome metal deficiency. The aim of this work was to identify genes and proteins differentially expressed by cells growing in low iron conditions in order to describe H. seropedicae response to iron limitation stress. For this purpose, and by using a transcriptomic approach, we searched and identified a set of genes up-regulated when iron was scarce. One of them, Hsero_2337, codes for a TonB-dependent transporter/transducer present in the serobactins biosynthesis genomic locus, with an unknown function. Another TonB-dependent receptor, the one encoded by Hsero_1277, and an inner membrane ferrous iron permease, coded by Hsero_2720, were also detected. By using a proteomic approach focused in membrane proteins, we identified the specific receptor for iron-serobactin internalization SbtR and two non-characterized TonB-dependent receptors (coded by genes Hsero_1277 and Hsero_3255). We constructed mutants on some of the identified genes and characterized them by in vitro growth, biofilm formation, and interaction with rice plants. Characterization of mutants in gene Hsero_2337 showed that the TonB-dependent receptor coded by this gene has a regulatory role in the biosynthesis of serobactins, probably by interacting with the alternative sigma factor PfrI, coded by gene Hsero_2338. Plant colonization of the mutant strains was not affected, since the mutant strain normally colonize the root and aerial part of rice plants. These results suggest that the strategies used by H. seropedicae to acquire iron inside plants are far more diverse than the ones characterized in this work. In vivo expression studies or colonization competition experiments between the different mutant strains could help us in future works to determine the relative importance of the different iron acquisition systems in the interaction of H. seropedicae with rice plants.

Proteomics-based identification of differentially abundant proteins reveals adaptation mechanisms of Xanthomonas citri subsp. citri during Citrus sinensis infection.

BACKGROUND: Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. A proteomic analysis under in planta infectious and non-infectious conditions was conducted in order to increase our knowledge about the adaptive process of Xac during infection. RESULTS: For that, a 2D-based proteomic analysis of Xac at 1, 3 and 5 days after inoculation, in comparison to Xac growth in NB media was carried out and followed by MALDI-TOF-TOF identification of 124 unique differentially abundant proteins. Among them, 79 correspond to up-regulated proteins in at least one of the three stages of infection. Our results indicate an important role of proteins related to biofilm synthesis, lipopolysaccharides biosynthesis, and iron uptake and metabolism as possible modulators of plant innate immunity, and revealed an intricate network of proteins involved in reactive oxygen species adaptation during Plants` Oxidative Burst response. We also identified proteins previously unknown to be involved in Xac-Citrus interaction, including the hypothetical protein XAC3981. A mutant strain for this gene has proved to be non-pathogenic in respect to classical symptoms of citrus canker induced in compatible plants. CONCLUSIONS: This is the first time that a protein repertoire is shown to be active and working in an integrated manner during the infection process in a compatible host, pointing to an elaborate mechanism for adaptation of Xac once inside the plant.

Iron isotope fingerprints of redox and biogeochemical cycling in the soil-water-rice plant system of a paddy field.

The iron isotope composition was used to investigate dissimilatory iron reduction (DIR) processes in an iron-rich waterlogged paddy soil, the iron uptake strategies of plants and its translocation in the different parts of the rice plant along its growth. Fe concentration and isotope composition (δ56Fe) in irrigation water, precipitates from irrigation water, soil, pore water solution at different depths under the surface water, iron plaque on rice roots, rice roots, stems, leaves and grains were measured. Over the 8.5-10cm of the vertical profiles investigated, the iron pore water concentration (0.01 to 24.3mg·l-1) and δ56Fe (-0.80 to -3.40‰) varied over a large range. The significant linear co-variation between Ln[Fe] and δ56Fe suggests an apparent Rayleigh-type behavior of the DIR processes. An average net fractionation factor between the pore water and the soil substrate of Δ56Fe≈-1.15‰ was obtained, taking the average of all the δ56Fe values weighted by the amount of Fe for each sample. These results provide a robust field study confirmation of the conceptual model of Crosby et al. (2005, 2007) for interpreting the iron isotope fractionation observed during DIR, established from a series of laboratories experiments. In addition, the strong enrichment of heavy Fe isotope measured in the root relative to the soil solution suggest that the iron uptake by roots is more likely supplied by iron from plaque and not from the plant-available iron in the pore water. Opposite to what was previously observed for plants following strategy II for iron uptake from soils, an iron isotope fractionation factor of -0.9‰ was found from the roots to the rice grains, pointing to isotope fractionation during rice plant growth. All these features highlight the insights iron isotope composition provides into the biogeochemical Fe cycling in the soil-water-rice plant systems studied in nature.

Iron acquisition and siderophore production in the fish pathogen Renibacterium salmoninarum.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767T type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330 µm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 µm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 µm), it did facilitate growth at lower iron concentrations (10 µm and 1 µm). An exception was the H-2 isolate, which only grew with 100 µm of iron sulphide. In turn, 100 µm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209T genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.

Paracoccidioides spp. ferrous and ferric iron assimilation pathways.

Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, (59)Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation.

The uptake of different iron salts by the yeast Saccharomyces cerevisiae.

Yeasts can be enriched with microelements, including iron; however, special physicochemical conditions are required to formulate a culture media that promotes both yeast growth and iron uptake. Different iron sources do not affect biomass formation; however, considering efficacy, cost, stability, and compatibility with Saccharomyces cerevisiae metabolism, ferrous sulphate is recommended.

The uptake of different iron salts by the yeast Saccharomyces cerevisiae

Yeasts can be enriched with microelements, including iron; however, special physicochemical conditions are required to formulate a culture media that promotes both yeast growth and iron uptake. Different iron sources do not affect biomass formation; however, considering efficacy, cost, stability, and compatibility with Saccharomyces cerevisiae metabolism, ferrous sulphate is recommended.

The uptake of different iron salts by the yeast Saccharomyces cerevisiae

Yeasts can be enriched with microelements, including iron; however, special physicochemical conditions are required to formulate a culture media that promotes both yeast growth and iron uptake. Different iron sources do not affect biomass formation; however, considering efficacy, cost, stability, and compatibility with Saccharomyces cerevisiae metabolism, ferrous sulphate is recommended.

Carotenoids, but not vitamin A, improve iron uptake and ferritin synthesis by Caco-2 cells from ferrous fumarate and NaFe-EDTA.

Due to the high prevalence of iron and vitamin A deficiencies and to the controversy about the role of vitamin A and carotenoids in iron absorption, the objectives of this study were to evaluate the following: (1) the effect of a molar excess of vitamin A as well as the role of tannic acid on iron uptake by Caco-2 cells; (2) iron uptake and ferritin synthesis in presence of carotenoids without pro-vitamin A activity: lycopene, lutein, and zeaxantin; and (3) iron uptake and ferritin synthesis from ferrous fumarate and NaFe-EDTA. Cells were incubated 1 h at 37 °C in PBS pH 5.5, containing (59) Fe and different iron compounds. Vitamin A, ferrous fumarate, ß-carotene, lycopene, lutein, zeaxantin, and tannic acid were added to evaluate uptake. Ferritin synthesis was measured 24 h after uptake experiments. Vitamin A had no effect on iron uptake by Caco-2 cells, and was significantly lower from NaFe-EDTA than from ferrous fumarate (15.2 ± 2.5 compared with 52.5 ± 8.3 pmol Fe/mg cell protein, respectively). Carotenoids increase uptake up to 50% from fumarate and up to 300% from NaFe-EDTA, since absorption from this compound is low when administered alone. We conclude the following: (1) There was no effect of vitamin A on iron uptake and ferritin synthesis by Caco-2cells. (2) Carotenoids significantly increased iron uptake from ferrous fumarate and NaFe-EDTA, and were capable of partially overcoming the inhibition produced by tannic acid. (3) Iron uptake by Caco-2 cell from NaFe-EDTA was significantly lower compared to other iron compounds, although carotenoids increased and tannic acid inhibited iron uptake comparably to ferrous fumarate.

Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.

A proteína de membrane externa IutA (iron uptake transport) é o receptor para aerobactina férrica, um fator de virulência encontrado mais frequentemente entre as amostras de E. coli pathogênicas para aves (APEC) do que entre os isolados fecais de aves saudáveis. O gene iutA da amostra APEC 9, sorotipo O2:H9, foi amplificado e clonado no vetor pET101/D-TOPO. O gene iutA 2.2 Kb foi sequenciado (AY602767) e mostrou alta similaridade para gene iutA de três plasmidios, dois da APEC, pAPEC-02-ColV (AY545598.4) e pTJ100 (AY553855.1), e um da amostra E. coli invasiva humana, pColV K30. A proteína IutA recombinante (r-IutA) foi produzida em Escherichia coli BL21(DE-3), solubilizada com uréia e purificada em coluna de níquel Ni-NTA. A r-IutA tem aproximadamente 74kDa e foi utilizada para produzir anticorpos anti-IutA. Este anticorpo reagiu com a r- IutA e com IutA da APEC13, como demonstrado por Western blot, mostrando que a r-IutA tem epitopos conservados e sua antigenicidade foi preservada. O anticorpo anti-IutA foi capaz de inibir a atividade biológica da IutA, inibindo o teste positivo de sensibilidade à cloacina DF13 apresentada pela APEC 9, contudo não inibiu o crescimento da APEC9 crescida em M9 e não protegeu os pintinhos inoculados com APEC 9, sugerindo que a APEC possui outro mecanismo de captação de íons ferro distinto da aerobactina.

The iron uptake mechanisms of enteroinvasive Escherichia coli

Enteroinvasive Escherichia coli strains (EIEC) of different serotypes isolated from patients with acute diarrhea were examined for the ability to produce siderophores and iron-regulated outer membrane proteins (IROMP). For iron starvation cultures were grown at 37°C in LB supplied with 200 muM of FONT FACE="Symbol">a /font>-alphadypirydil. All strains produced enterobactin and twelve (40%) produced aerobactin. The strains showed IROMP varying from 67-82 kDa. Proteins were either induced or stimulated by the iron starvation. Differences were observed in the electrophoretic profile among the serotypes, originating 5 electrophoretic profiles. All serotypes expressed proteins of 82 kDa (FepA) and 76 kDa (IutA) (except serotype O28ac:H- that did not produce the 76 kDa protein). Several strains (O29:H-, O144:H-, O152:H-, and O167:H-) expressed IutA in the outer membrane, in the absence of aerobactin production. Additionally to well characterized proteins (FepA and IutA), we found two IROMP of unknown function in some serotypes: a 71 kDa protein was detected in three profiles and a 67 kDa protein was present in serotype O152:H-. Moreover, two bands (39 and 43 kDa) which were not iron-regulated bound specifically to human lactoferrin.

Cepas de Escherichia coli enteroinvasora de diferentes sorotipos isoladas de pacientes com diarréia aguda foram examinadas quanto a capacidade de produzir sideróforos e proteínas de membrana externa reguladas pelo ferro (IROMP). O crescimento bacteriano em meio com deficiência em Fe foi obtido em caldo Lúria acrescido de 200 mM de alfa- FONT FACE="Symbol">a /font>dipiridil. Todas as cepas produziram enterobactina e 40% produziram aerobactina. As cepas produziram IROMPs com MM variando de 82-67 kDa. As proteínas foram induzidas ou estimuladas pela deficiência de ferro. Diferenças foram observadas no perfil eletroforético entre os sorotipos, originando 5 perfis eletroforéticos. Todos os sorotipos, com exceção do sorotipo O28ac:H- (onde a proteína de 76 kDa não foi produzida), expressaram proteínas de 82 kDa (FepA) e 76 kDa (IutA). Várias cepas (O29:H-, O144:H-, O152:H- e O167:H-) expressaram IutA na membrana externa, na ausência da produção de aerobactina. Além das proteínas (FepA e IutA), foram encontradas em alguns sorotipos duas IROMPs de função desconhecida: uma proteína de 71 kDa foi detectada em 3 perfis e uma de 67 kDa presente no sorotipo O152:H-. Além disso, 2 bandas (39 e 43 kDa), as quais não foram reguladas pelo ferro, mostraram afinidade à lactoferrina humana.