Summary

The main objective of this line of research is the development of new treatments and diagnostics based on Fe(III) uptake mechanisms for infectious diseases in aquaculture fish caused by pathogenic Gram-negative bacteria.
Our approach consists of harnessing bacterial iron(III) uptake mechanisms based on the use of siderophores. Iron is an essential element for growth but with very limited availability due to its low solubility at physiological pH. Siderophores are low-molecular-weight compounds that, when secreted into the surrounding environment, effectively chelate iron(III) and then specifically transport it through specific membrane protein receptors into the cells, where they release it.
Our work primarily involves the isolation, structural characterization, and synthesis of siderophores and some of their analogues responsible for Fe(III) uptake in each pathogenic bacterium. Additionally, the biosynthesis of the isolated siderophores is studied, identifying the genes involved and their assembly. The proteins involved not only in their biosynthesis but also in the uptake and transport of the ferric complex of the siderophore are also studied. The main biotechnological applications to be developed with this acquired knowledge focus on:

• The development of new antimicrobials by conjugating the siderophore analogue with a known antibiotic (Trojan Horse strategy)

• The preparation of fluorescent probes as new diagnostic tools with their conjugation with a known fluorophore.

• The development of new vaccines using the outer membrane proteins of the iron (III) siderophore complex as antigens. For more detailed information on this application, see the research line titled PRODUCTION AND ISOLATION OF RECOMBINANT PROTEINS

We are investigating three of the most important infectious diseases in aquaculture: vibriosis, photobacteriosis (pasteurellosis), and furunculosis, caused by the three Gram-negative pathogens: Vibrio anguillarum, Photobacterium damselae subsp. piscicida, and Aeromonas salmonicida subsp. salmonicida, respectively.

Vibriosis

Caused by marine bacteria belonging to Vibrio anguillarum (Va), we have successfully isolated and characterized two of the three iron transport systems of this bacterium, which were named vanchrobactin and piscibactin (the minority anguibactin system was described several years earlier and is only present in Va serotype O1 strains that carry the pJM1 plasmid).

Vanchrobactin transport system: We isolated and characterized this siderophore for the first time from the Va RV22 serotype O2 strain. Its total synthesis was achieved, along with that of several active analogues, which were used in the preparation of new antimicrobial agents following the so-called Trojan Horse strategy. These studies identified FvtA as the transporter protein of the vanchrobactin iron complex. Its iron complexes were studied using spectrophotometric and potentiometric methods.

Piscibactin transport system: It was previously isolated and identified from Photobacterium damselae subsp. piscicida but was subsequently found to be widely distributed in bacteria of the Vibrionaceae family. Additional studies in Vibrio anguillarum showed that piscibactin contributed more than vanchrobactin to the virulence of bacterial infection in sole. More recently, we isolated and identified in Va RV22 the presence of photoxenobactin E, which has a terminal thiocarboxylic acid function, and we have shown that its gallium complex is in equilibrium between its two tautomeric forms where Ga³⁺ is coordinated through either the sulfur atom or the thiocarboxylate oxygen group.

Fotobacteriosis (pasteurellosis)

By analyzing the genetic cluster involved in the biosynthesis of the siderophore biosynthesized by Photobacterium damselae subsp. piscicida, responsible for photobacteriosis, we were able to predict, isolate, and structurally characterize piscibactin, as well as prepiscibactin as an intermediate involved in its biosynthesis. A possible biosynthetic pathway was proposed, linking the deduced structure to the genes and enzymes involved in its assembly. The total synthesis of prepiscibactin and piscibactin was carried out. The synthesis of various piscibactin analogs allowed us to establish the importance of the configuration of the hydroxyl group at the C13 position both for chelating iron or gallium and for the presence of siderophore activity.

FrpA was identified as the outer membrane protein transporting the piscibactin ferric complex. Its subsequent cloning, expression, and purification in E. coli led to the production of rFrpA, which was successfully tested as a vaccine against photobacteriosis. Its three-dimensional structure was approximated by crystal X-ray diffraction.

The transmission of plasmid pPHDP70, which encodes piscibactin biosynthesis, by conjugation from P. damselae subsp. piscicida to a mutant strain of the mollusc-pathogenic Gram-negative bacterium Vibrio alginolyticus, which had been eliminated from its ability to produce its siderophore vibrioferrin, allowed not only the restoration of growth of the mutant strain of V. alginolyticus under iron-deficient conditions, but also the detection of piscibactin production. The horizontal transfer of plasmid pPHDP70 demonstrates the ease with which new iron uptake mechanisms can be transmitted between bacteria, with the consequent resurgence of new pathogens in the marine environment.

Furunculosis

Through genome mining of the genome of a strain of Aeromonas salmonicida subsp. salmonicida, the causative agent of this infectious disease, two types of gene clusters were identified that are involved in the biosynthesis of two different classes of siderophores. This analysis first allowed us to predict, then isolate and structurally characterize, the two transport systems of this bacterium: amonabactins and acinetobactin.

Acinetobactin transport system: This compound was previously identified as the siderophore involved in Fe(III) uptake by the human pathogen Acinetobacter baumanii. The total synthesis of the acinetobactin enantiomer, as well as various analogues, allowed us not only to deduce important siderophore structure-activity relationships but also to identify a simplified analogue that can be used as a vector in the preparation of conjugates. These studies also allowed us to identify FstA as the transport protein of the acinetobactin ferric complex.

Sistema de transporte acinetobactina: Este compuesto fue previamente identificado como el sideróforo implicado en la captación de Fe(III) de la bacteria patógena en humanos Acinetobacter baumanii. La síntesis total del enantiómero de acinetobactina así como de diversos análogos permitieron no solo deducir importantes relaciones de estructura-actividad siderófora sino también identificar un análogo simplificado que puede ser utilizado como vector en la preparación de conjugados. Dichos estudios permitieron además identificar FstA como la proteína transportadora del complejo férrico de acinetobactina.

Acinetobactin transport system: This compound was previously identified as the siderophore involved in Fe(III) uptake by the human pathogen Acinetobacter baumanii. The total synthesis of the acinetobactin enantiomer and several analogues not only allowed us to deduce important siderophore structure-activity relationships but also to identify a simplified analogue that can be used as a vector in the preparation of conjugates. These studies also allowed us to identify FstA as the transport protein for the acinetobactin iron complex.

The four forms of amonabactin previously identified as siderophores involved in Fe(III) uptake by the human pathogen Aeromonas hydrophila were isolated and identified. We designed a more efficient total synthesis of this type of siderophore. The preparation and biological assay of various amonabactin derivatives allowed us to identify a simplified analogue that could be used as a vector for the preparation of conjugates. This simplified amonabactin analogue was used as a vector to prepare a fluorescent probe with the fluorophore sulforhodamine B. This probe was also used to determine the entry route of Aeromonas salmonicida subsp. salmonicida labeled with this probe into zebrafish. These studies also allowed us to identify FstC as the amonabactin receptor protein, whose recombinant protein rFstC was produced and tested as a vaccine against furunculosis.

Additionally, we have studied the siderophore-mediated iron uptake systems of other pathogenic bacteria: Vibrioferrin in Edwardsiella tarda, the agent responsible for edwardsiellosis, acinteobactin and fimsbactins in Acinetobacter baumanii, citrate and vibrioferrin in Photobacterium damselae subsp. damselae, and amphibactins and piscibactin in Vibrio neptunius.