Estudio químico de bacterias de origen marinoaislamiento de productos naturales bioactivos, proteínas recombinantes receptoras de sideróforos de bacterias patógenas en peces de acuicultura y síntesis de conjugados de sideróforos con nanopartículas magnéticas

Abstract

This Doctoral Thesis is divided into three parts. The first part describes the importance of iron uptake systems in fish pathogenic bacteria and the use of siderophore-receptor proteins as potential vaccine candidates. In the second part, the synthesis of a conjugate of feroxamine siderophore and magnetic nanoparticles is shown as an attempt to create a new surface able to capture Yersinia enterocolitica WC-A. Finally, in chapter III the structural and stereochemical determination of the tetrapeptide cereusitin A is described as well as the detection and identification of the piscibactin-Ga(III) complex in the supernatants of the cultures of Vibrio anguillarum RV22 and its vabF mutant. Furthermore, the piscibactin is shown as an important molecule in the iron uptake system and the virulence of V. aguillarum. Chapter I This chapter discusses the importance of aquaculture and the need to develop new treatments for the control of infections produced by pathogenic bacteria. In addition, the mechanism of iron uptake along with the advantages of using siderophores receptor outer membrane proteins as vaccines candidates is described broadly. Here are presented the methodology of cloning, expression and purification of the recombinant proteins FvtA (receptor of the siderophore vancrobactin produced by Vibrio anguillarum RV22) and FstC (receptor of the siderophores amonabactin biosynthesized by Aeromonas salmonicida subsp. salmonicida VT45). Additionally, the expression and purification of rFstB protein (acinetobactin receptor produced by A. salmonicida subsp. salmonicida VT45) is shown. Finally, the results obtained when testing rFvtA, rFstB and rFstC as a vaccine in flounders infected with vibriosis and forunculosis are presented. Chapter II This chapter is dedicated to the application of siderophores and magnetic nanoparticles to the study of a bacterial detection mechanism. The applications of siderophores in different fields are described along with the design process of magnetic nanomaterials and its versatility and capability in the conjugate synthesis to develop applications. The synthesis of the conjugate of feroxamine and functionalized magnetic nanoparticles (MNP@SiO2@NH@Fa), its characterization and capture capacity towards Yersinia enterocolitica WC-A are described. Also, the synthesis of an enterobactin analogue that can be used for the synthesis of conjugates is shown. Chapter III In this chapter is made a brief introduction of the importance of marine microorganisms as producers of natural products, their biological activity and their great structural diversity. Also, the most general methods for the selection of culture conditions in the obtainment of compounds mainly produced by bacteria are exposed. The structural elucidation process of the tetrapeptide cereusitin A produced by Bacillus cereus RKHC-09 is shown by mono and bidimensional nuclear magnetic resonance experiments as well as low and high resolution mass spectrometry analysis. Additionally, absolute stereochemistry was determined by means of the Marfey’s method. Finally, here is shown the procedure developed for the detection of piscibactin- Ga(III) complex from culture supernatant of V. anguillarum RV22 and its vabF mutant by liquid chromatography coupled to mass spectrometry (LC-MS).