Miguel Balado - Academia.edu (original) (raw)

Papers by Miguel Balado

Journal of Inorganic Biochemistry, 2022

Amonabactins, the siderophores produced by some pathogenic bacteria belonging to Aeromonas genus,... more Amonabactins, the siderophores produced by some pathogenic bacteria belonging to Aeromonas genus, can be used for the preparation of conjugates to be imported into the cell using their specific transport machinery. Herein, we report the design and synthesis of a new amonabactin-based fluorescent probe by conjugation of the appropriate amonabactin analogue to sulforhodamine B (AMB-SRB) using a thiol-maleimide click reaction. Growth promotion assays and fluorescence microscopy studies demonstrated that the AMB-SRB fluorescent probe was able to label the fish pathogenic bacterium A. salmonicida subsp. salmonicida through its outer membrane transport (OMT) protein FstC. The labelling of other Aeromonas species, such as the human pathogen A. hydrophila, indicates that this probe can be a very useful molecular tool for studying the amonabactin-dependent iron uptake mechanism. Furthermore, the selective labelling of A. salmonicida and other Aeromonas species in presence of other fish pathogenic bacteria, suggest the potential application of this probe for detection of Aeromonas in water and other fish farming samples through fluorescence assays.

Frontiers in Microbiology, 2021

The high-pathogenicity island irp-HPI is widespread among Vibrionaceae encoding the piscibactin s... more The high-pathogenicity island irp-HPI is widespread among Vibrionaceae encoding the piscibactin siderophore system. The expression of piscibactin genes in the fish pathogen Vibrio anguillarum is favored by low temperatures. However, information about the regulatory mechanism behind irp-HPI gene expression is scarce. In this work, in-frame deletion mutants of V. anguillarum defective in the putative regulators AraC1 and AraC2, encoded by irp-HPI, and in the global regulators H-NS and ToxRS, were constructed and their effect on irp-HPI gene expression was analyzed at 15 and 25°C. The results proved that only AraC1 (renamed as PbtA) is required for the expression of piscibactin biosynthesis and transport genes. PbtA inactivation led to an inability to grow under iron restriction, a loss of the outer membrane piscibactin transporter FrpA, and a significant decrease in virulence for fish. Inactivation of the global repressor H-NS, which is involved in silencing of horizontally acquired g...

Fish & Shellfish Immunology, 2019

Photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) remains one of the main... more Photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) remains one of the main infectious diseases affecting cultured fish in Mediterranean countries. Diverse vaccine formulations based in the use of inactivated bacterial cells have been used with unsatisfactory results, especially in newly cultured species like sole (Solea senegalensis). In this work, we describe the use of the outer membrane receptor (FrpA) of the siderophore piscibactin produced by Pdp as a novel subunit vaccine against photobacteriosis. FrpA has been cloned and expressed in Escherichia coli under an arabinose-inducible promoter. A recombinant protein (rFrpA) containing the pelB localization signal and a His tag was constructed to obtain a pure native form of the protein from E. coli outer membranes. The immunogenicity of rFrpA, and its protective effect against photobacteriosis, was tested by i.p. injection of 30 μg of the protein, mixed with Freund's adjuvant, in sole fingerlings with two immunizations separated by 30 days. Results showed that using either pure rFrpA or whole cells as immobilized antigens in ELISA assays, rFrpA induces the production of specific antibodies in sole. An experimental infection using fish vaccinated with rFrpA or formalin-killed whole cells of Pdp showed that both groups were protected against Pdp infection at similar levels, with no significant differences, reaching RPS values of 73% and 79%, respectively. Thus, FrpA constitutes a promising antigen candidate for the development of novel more effective vaccines against fish photobacteriosis.

Journal of Applied Microbiology, 2019

Aims: Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photo... more Aims: Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photobacterium malacitanum and Photobacterium andalusiense associated with diseases of cultured redbanded seabream (Pagrus auriga) were studied. The exoenzymatic activities, adherence and cytotoxic capabilities, and iron-uptake mechanisms were determined both in bacterial extracellular products (ECP) and whole bacterial cells. The histopathology damages provoked on redbanded seabream by the ECP was also studied. Methods and Results: The highest exoenzymatic activities of the ECP were alkaline-and acid-phosphatase, phosphohydrolase and lipase. The ECP were strongly lethal for fish at 4-96 h post-inoculation (p.i). Histological changes were evident at 96 hpi of ECP, affecting head kidney, splenic parenchyma and heart. Cytotoxicity assays, on three fish lines and one human cell line, were conducted using whole bacterial cells and their ECP. The new species tested were cytotoxic only for fish cell lines using whole bacterial cells. Bacterial adherence showed an adherence index moderate on CHSE-214 cell line. All strains showed variable haemolytic activity, and were able to grow under ironlimiting conditions, although the CAS reactivitiy was very low. However, all strains produced high amounts of extracelullar citrate that could be used as iron carrier, and use haem as iron source, except the P. toruni strains because a deletion in the genomic region encoding this ability in all Vibrionaceae members. Conclusions: The toxic activity of the bacterial ECPs was thermolabile, and not associated with their thermoresistant lipopolysaccharide content. The virulence of the strains tested could not be related to the haemolytic activity. Iron uptake could be based on the use of endogenous citrate as iron carrier and P. toruni lacks the ability to use haem as iron source. Significance and Impact of the study: The study analyses for the first time the virulence properties of three new species of Photobacterium pathogenic for fish.

Advances in Polymer Technology, 2018

In this research, iron oxide magnetic nanoparticles (NPs) were synthesized by chemical co-precipi... more In this research, iron oxide magnetic nanoparticles (NPs) were synthesized by chemical co-precipitation method. The surface of particles was grafted with βcyclodextrin/N,N-dimethylacrylamide (β-CD/DMAA) copolymer. Potency of NPs as carrier of antibiotic ofloxacin (OFL) was evaluated. They were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, elemental analysis, and thermogravimetric analysis. The effect of the adsorption parameters such as pH, contact time, and temperature for sorption of drug onto NPs was evaluated and optimized. The drug release profile showed that approximately 60% of the adsorbed drug was released in the first 10 min at pH of 1.2 (simulated gastric fluid) and 55% was released within 30 hr in pH of 7.4 (simulated intestinal fluid). Findings of study suggested the suitability of β-CD/DMAA grafted magnetic NPs for OFL delivery.

Food Chemistry, 2018

The large variety of fish formats which are globally commercialized supports use of meta-evaluati... more The large variety of fish formats which are globally commercialized supports use of meta-evaluation studies to test discrimination power among molecular keys available for traceability of highlydegraded and/or chemically-modified DNA material. This paper shows that a combination of DNA identification methods validated for genus Merluccius allows 100% species assignment in hake products and offers higher diagnostic power (97% on products) than individual methods, i.e. Hake-ITS1-RFLP (89%) or Hake-Cytochrome b-RFLP (83%). A global 31% product mislabelling involved 15% of products affected by internal species substitution, as the main cause of mislabelling in ultrafrozen products, and another 16% affected by external species substitution with non-hake species, as the cause of mislabelling in processed and cooked hake-based products. The combination of both DNA keys minimizes the tool-associated rate of diagnostic failure, allowing decoupling of mislabelling categories and maximizing the quantitative adscription of products to species on any kind of hake-based products.

Veterinary microbiology, 2017

Photobacterium damselae subsp. damselae (Pdd) is a marine bacterium that can infect numerous spec... more Photobacterium damselae subsp. damselae (Pdd) is a marine bacterium that can infect numerous species of marine fish as well as other species including humans. Low iron availability is one of the signs that bacterial pathogens can detect in order to begin colonizing their host, and the reduction of iron levels is a nonspecific host defense strategy that prevents bacterial proliferation. In this work a proteomic approach was used to study the gene expression adaptations of a Pdd strain in response to iron availability. A comparative analysis of induced proteins in both high- and low-iron conditions showed profound cellular metabolic adaptations that result, for instance, in amino acid requirement. It also provided important information about the changes that occur in the energetic metabolism induced by the surrounding iron levels, allowing for the identification of novel potential virulence factors. Among others, genes involved in the synthesis and transport of a vibrioferrin-like sid...

Journal of Separation Science, 2009

A new solid-phase extraction coupled with magnetic carrier technology was developed for extractio... more A new solid-phase extraction coupled with magnetic carrier technology was developed for extraction of bisphenol A (BPA) and diethylstilbestrol (DES) from water samples. The SPE sorbents, functionalized magnetic nanoparticles (Fe 3 O 4 @SiO 2 /b-CD, core/shell), were synthesized in a two-stage system. The material was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and a vibrating sample magnetometer. SPE extraction parameters, such as volume and pH of sample, adsorption time, and desorption conditions were optimized. Under selected conditions: 250 mL of water sample, 0.1 g of sorbents and elution with methanol (3 mL with 1% acetic acid), the extraction was completed in 25 min. SPE followed by HPLC was employed to determine BPA and DES in environmental samples. The developed method provided spiked recoveries of 80-105%, relative standard deviations of less than 7%, and LOD of BPA (20.0 ng/L) and DES (23.0 ng/L), respectively. The proposed method offered easy preparation of sorbents, rapid analysis, high enrichment yields, and reliable quantitative assay.

Journal of Sea Research, 2014

Population genetic data of marine genetic resources add up to establish their pattern of connecti... more Population genetic data of marine genetic resources add up to establish their pattern of connectivity. Such pattern determines the choice of a suitable management strategy to those resources in space and time. This study addresses the pattern of connectivity among European hake grounds upon the largest sampling effort so far developed in this species. Bayesian inference made on multilocus genotypic data provides evidence that a large genetic connectivity exists among Atlantic grounds and is mediated by significant migration rates stepping up from the Celtic Sea towards its adjacent Atlantic grounds. Therefore, the spawning biomass of the northern hake population could play a crucial role at ensuring the sustainability of southern hake fish grounds. The deepest restriction to gene flow was observed at the easternmost side of the Alboran Sea what suggests that the Almeria-Oran Oceanographic Front is an effective barrier keeping apart the two major gene pools so far shown in this species. A molecular clock calibrated for cytochrome b sequences of fishes and applied to the divergence between hake pools (0.3%) suggests that the Atlantic-Mediterranean population split might date back to the Middle Pleistocene (150,000 years BP). Complementarily, Bayesian treatments of multilocus genotypes indicate that such barrier to gene flow remains active nowadays. This study shows the upmost interest of future integrative efforts to incorporate population genetic data into current assessment practices, in order to better manage European hake populations and make its exploitation sustainable.

Journal of Aquatic Food Product Technology, 2004

The application of molecular genetics to the identification of fishes is an appropriate technolog... more The application of molecular genetics to the identification of fishes is an appropriate technology for quality control and commercial tracking. We have applied a DNA-based methodology to identify the exact species of Argentinean hake included in commercial products. The first test consisted in the PCR amplification of a 122 bp fragment from the cytochrome bgene to assess the presence of DNA from hakes. This test being positive, it was followed by a second test consisting of the amplification and digestion of a PCR fragment of 464 bp from the cytochrome bgene, to determine the identity of the commercialized species. Since a single restriction enzyme provided full diagnosis of the southern Atlantic hake species present in the samples, this methodology would be of great help in traceability across the food-chain that comprises (1) the authentication of Argentinean hake products, (2) the control of fraud to consumers, (3) the industrial advice regarding the labeling of commercial products, and (4) the fishery forensics of Argentinean hakes.

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, Oct 5, 2017

Photobacterium damselae subsp. piscicida (Pdp) is an intracellular fish pathogen that causes phot... more Photobacterium damselae subsp. piscicida (Pdp) is an intracellular fish pathogen that causes photobacteriosis, a disease proven deadly in farmed fish worldwide. This work focuses on the analysis of genome sequences, chromosomes structure and gene contents of two strains from Sparus aurata (DI21) and Solea senegalensis (L091106-03H), isolated on the Spanish Atlantic coast. The comparative genomic analysis revealed that DI21 and L091106-03H share 98% of their genomes, including two virulence plasmids: pPHDP70 encoding siderophore piscibactin synthesis and pPHDP10 encoding the apoptotic toxin AIP56. Both genomes harbour a surprisingly large number of IS elements accounting for 12-17% of the total genome, representing an IS density of 0.15 elements per kb, one of the highest IS density values in a bacterial pathogen. This massive proliferation of ISs is responsible for the generation of a high number of pseudogenes that caused extensive loss of biological functions. Pseudogene formation...

Microorganisms, 2021

Bismuth is a heavy metal with antibacterial properties that has a long history of medicinal use. ... more Bismuth is a heavy metal with antibacterial properties that has a long history of medicinal use. The results reported here suggest that bismuth(III) (chelated with deferiprone) could be used in aquaculture systems to treat bacterial disease outbreaks, greatly reducing antibiotic use. We tested bismuth susceptibility in a collection of aquaculture bacterial pathogens. In the presence of bismuth concentrations ranging from 1.3 to 13 µM, most bacteria started showing a drastic decrease in their growth ability, although with high inter- and intraspecific variability. The minimal inhibitory concentrations of bismuth ranged from 13 to more than 780 µM, depending on bacterial species and strain. The results of in vivo assays suggest that low concentrations of bismuth could be especially effective to treat vibriosis caused by Vibrio anguillarum, since bismuth greatly reduced mortality in experimentally infected fish without any observable side effects. A bismuth therapy, alone or combined w...

JBIC Journal of Biological Inorganic Chemistry, 2021

Piscibactin (Pcb) is a labile siderophore widespread among Vibrionaceae. Its production is a majo... more Piscibactin (Pcb) is a labile siderophore widespread among Vibrionaceae. Its production is a major virulence factor of some fish pathogens such as Photobacterium damselae subsp. piscicida and Vibrio anguillarum. Although FrpA was previously suggested as the putative outer membrane transporter (OMT) for ferri-piscibactin, its role in piscibactin uptake was never demonstrated. In this work, we generated mutants of V. anguillarum defective in FrpA and analyzed their ability to use piscibactin as iron source. The results showed that inactivation of frpA completely disables piscibactin utilization, and the original phenotype could be restored by gene complementation, confirming that FrpA is the OMT that mediates ferri-Pcb uptake. Additionally, the ability of several Pcb thiazole analogues, with different configurations at positions 9, 10, and 13, to be internalized through FrpA, was evaluated measuring their ability to promote growth under iron deficiency of several indicator strains. Th...

Frontiers in Cellular and Infection Microbiology, 2021

Vibrio neptunius is an inhabitant of mollusc microbiota and an opportunistic pathogen causing dis... more Vibrio neptunius is an inhabitant of mollusc microbiota and an opportunistic pathogen causing disease outbreaks in marine bivalve mollusc species including oysters and clams. Virulence of mollusc pathogenic vibrios is mainly associated with the production of extracellular products. However, siderophore production is a common feature in pathogenic marine bacteria but its role in fitness and virulence of mollusc pathogens remains unknown. We previously found that V. neptunius produces amphibactin, one of the most abundant siderophores in marine microbes. In this work, synthesis of the siderophore piscibactin was identified as the second siderophore produced by V. neptunius. Single and double mutants in biosynthetic genes of each siderophore system, piscibactin and amphibactin, were constructed in V. neptunius and their role in growth ability and virulence was characterized. Although the High Pathogenicity Island encoding piscibactin is a major virulence factor in vibrios pathogenic fo...

Antibiotics, 2021

Vibrio neptunius is an important pathogen of bivalve mollusks worldwide. Several metalloproteases... more Vibrio neptunius is an important pathogen of bivalve mollusks worldwide. Several metalloproteases have been described as virulence factors in species of Vibrio that are pathogenic to bivalves, but little is known about the contribution of these potential virulence factors to Vibrio neptunius pathogenesis. In silico analysis of the genome of V. neptunius strain PP-145.98 led to the identification of two hitherto uncharacterized chromosomal loci encoding a probable vibriolysin-like metalloprotease and a putative collagenase, which were designated VnpA and ColA, respectively. Single defective mutants of each gene were obtained in V. neptunius PP-145.98, and the phospholipase, esterase and collagenase activities were studied and compared with those of the wild-type strain. The results showed that the single inactivation of vnpA resulted in a 3-fold reduction in phospholipase/esterase activity. Inactivation of colA reduced the collagenase activity by 50%. Finally, infection challenges pe...

Environmental Microbiology, 2020

Amphiphilic siderophores including amphibactins are the most abundant siderophores in oceans. Gen... more Amphiphilic siderophores including amphibactins are the most abundant siderophores in oceans. Genes putatively encoding the amphibactin system were proposed in some bacteria and homologues of these genes are particulary abundant in multiple bacterial lineages inhabitant of low-iron seawater. However, since no defective mutant strains in any of these genes were studied to date, their role in amphibactin synthesis or uptake were not demonstrated. In this work, an in silico analysis of the genome of the mollusc pathogen V. neptunius lead us to identify a gene cluster (denoted absABDEF) that is predicted to encode an amphibactin-like siderophore and several mutant strains unable to synthesize or use siderophores were constructed. The results showed that genes absABDEF are required for amphibactin synthesis. Comparative chemical analysis of V. neptunius wild type and biosynthesis mutants allowed us to identify a mixture of nine amphibactin forms produced by this bacterium. In addition, gene abtA encode the ferri-amphibactin outer membrane transporter. The prevalence of the amphibactin system in bivalve hemolymph microbiota was also studied. We found that amphibactin system is widespread in hemolymph microbiota including both commensal and pathogenic bacterial species. Thus, its contribution to bacterial fitness must be more related to environmental persistence than to pathogenicity. This article is protected by copyright. All rights reserved.

ACS Infectious Diseases, 2019

Amonabactins are a group of four related catecholate siderophores produced by several species of ... more Amonabactins are a group of four related catecholate siderophores produced by several species of the genus Aeromonas, including A. hydrophila and the fish pathogen A. salmonicida subsp. salmonicida. Although the gene cluster encoding amonabactin biosynthesis also contains a gene that could encode the ferri-siderophore receptor (fstC), to date there is no experimental evidence to explain its role. In this work, we report the identification of the amonabactins' outer membrane receptor and the determination of the minimal structural parts of these siderophores involved in the molecular recognition by their cognate receptor. The four natural amonabactin forms (P750, T789, P693, and T732) and some mono and biscatecholate amonabactin analogues were chemically synthesized, and their siderophore activity on A. salmonicida FstC(+) and FstC(−) strains was evaluated. The results showed that each amonabactin form has quite different growth promotion activity, with P750 and T789 the most active. The outer membrane receptor FstC recognizes more efficiently biscatecholate siderophores in which the length of the linker between the two iron-binding catecholamide units is 15 atoms (P750 and T789) instead of 12 atoms (P693 and T732). Analysis of the siderophore activity of synthetic analogues indicated that the presence of Phe or Trp residues is not required for siderophore recognition. The results together point toward evidence that the amonabactin receptor FstC admits a high degree of ligand plasticity. We also showed that FstC is present in most Aeromonas species, including relevant human and animal pathogens as A. hydrophila. From the results obtained, we concluded that the ferri-amonabactin uptake pathway involving the outer membrane transporter FstC possesses a considerable functional plasticity that could be exploited for delivery of antimicrobial compounds into the cell. This would allow the use of the siderophore-based iron uptake mechanisms to combat infections caused by species of the genus Aeromonas.

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Frontiers in microbiology, 2018

causes vibriosis, a hemorrhagic septicaemia that affects many cultured marine fish species worldw... more causes vibriosis, a hemorrhagic septicaemia that affects many cultured marine fish species worldwide. Two catechol siderophores, vanchrobactin and anguibactin, were previously identified in this bacterium. While vanchrobactin is a chromosomally encoded system widespread in all pathogenic and environmental strains, anguibactin is a plasmid-encoded system restricted to serotype O1 strains. In this work, we have characterized, from a serotype O2 strain producing vanchrobactin, a novel genomic island containing a cluster of genes that would encode the synthesis of piscibactin, a siderophore firstly described in the fish pathogen subsp. . The chemical characterization of this siderophore confirmed that some strains of produce piscibactin. An analysis of the available genomes showed that this genomic island is present in many of the highly pathogenic strains lacking the anguibactin system. The construction of single and double biosynthetic mutants for vanchrobactin and piscibactin allowed...

Frontiers in cellular and infection microbiology, 2017

Photobacterium damselae subsp damselae (Pdd) is a Vibrionaceae that has a wide pathogenic potenti... more Photobacterium damselae subsp damselae (Pdd) is a Vibrionaceae that has a wide pathogenic potential against many marine animals and also against humans. Some strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there are virulent strains that do not produce vibrioferrin, but they still give a strong positive reaction in the CAS test for siderophore production. In an in silico search on the genome sequences of this type of strains we could not find any ORF which could be related to a siderophore system. To identify genes that could encode a siderophore-mediated iron acquisition system we used a mini-Tn10 transposon random mutagenesis approach. From more than 1,400 mutants examined, we could isolate a mutant (BP53) that showed a strong CAS reaction independently of the iron levels of the medium. In this mutant the transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase that participates in the tricarboxylic acid cycle. The ...