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[Resumen]: En los últimos años, la prevalencia de las enfermedades infecciosas producidas por patógenos y la resistencia bacteriana a los antibióticos han aumentado de forma considerable. Debido a que se están convirtiendo en una amenaza para la salud pública, se ha hecho necesario el desarrollo de nuevos agentes antimicrobianos. Por este motivo se ha planteado en este Trabajo de Fin de Grado la preparación de un sideróforo unido covalentemente a un conector, para su posterior conjugación con polímeros con el fin de ensayarlo como antimicrobiano. El sideróforo seleccionado para dirigir selectivamente el conjugado sintético sideróforo-polímero, fue un análogo de la amonabactina P750, que es uno de los sideróforos producido por la bacteria patógena Aeromonas salmonicida subsp salmonicida, mientras que el conector elegido fue un polietilenglicol (PEG-3). En el desarrollo de este trabajo se planteó en primer lugar un análisis retrosintético para la síntesis del sideróforo elegido acoplado al conector, en el que se seleccionaron tres subestructuras principales: una subestructura del sideróforo formada por una lisina y un catecol (S-7), otra subestructura del sideróforo constituida por una glicina, una lisina y un catecol (S-12) y el conector, que contiene tres unidades etilenglicol (C-13). A continuación, se consiguió sintetizar y caracterizar cada una de las subestructuras propuestas y se logaron conectar con éxito para obtener el sideróforo acoplado con el conector planteado (SC-16). Desafortunadamente, los ensayos de acoplamiento de los polímeros con los fragmentos del sideróforo sintetizados fueron infructuosos. A la vista de los resultados obtenidos y con el fin de conseguir preparar los conjugados de interés, se plantea variar las condiciones de reacción de acoplamiento y/o emplear otros polímeros funcionalizados. [Resumo]: Nos últimos anos, aumentou considerablemente a prevalencia de enfermidades infecciosas causadas por patóxenos e a resistencia bacteriana aos antibióticos. Debido a que se están a converter nunha ameaza para a saúde pública, fíxose necesario o desenvolvemento de novos axentes antimicrobianos. Por este motivo, neste Traballo Fin de Grao propúxose a elaboración dun sideróforo unido covalentemente a un conector, para a súa posterior conxugación con polímeros co fin de probalo como antimicrobiano. O sideróforo seleccionado, para dirixirse selectivamente ao conxugado sintético sideróforo-polímero, foi un análogo da amonabactina P750, que é un dos sideróforos producidos pola bacteria patóxena Aeromonas salmonicida susp. salmonicida, mentres que o conector elixido foi un polietilenglicol (PEG-3). No desenvolvemento deste traballo propúxose primeramente unha análise retrosintética para a síntese do sideróforo elixido acoplado ao conector, na que se seleccionaron tres subestruturas principais: unha subestrutura do sideróforo formada por unha lisina e un catecol (S-7), outra subestrutura do sideróforo formada por unha glicina, unha lisina e un catecol (S-12) e o conector, que contén tres unidades de etilenglicol (C-13). A continuación, foi posible sintetizar e caracterizar cada unha das subestruturas propostas e conectalas para obter o sideróforo acoplado ao conector proposto (SC-16). Desafortunadamente, as probas de acoplamento dos polímeros cos fragmentos de sideróforo sintetizados non tiveron éxito. Á vista dos resultados obtidos e para poder preparar os conxugados de interese, proponse variar as condicións da reacción de acoplamento e/ou empregar outros polímeros funcionalizados. [Abstract]: In recent years, the prevalence of infectious diseases caused by pathogens and bacteria resistant to antibiotics have increased considerably. Because they are becoming a threat to public health, the development of new antimicrobial agents has become necessary. For this reason, the preparation of a siderophore covalently bonded to a linker has been proposed in this project, for its subsequent conjugation with polymers in order to test it as an antimicrobial. The selected siderophore, that will act as a targeting ligand in the synthetic siderophore-polymer conjugate, was an analog of amonabactin P750, which is one of the siderophores produced by the pathogenic bacterium Aeromonas salmonicida susp. salmonicida, and the chosen linker was a polyethylene glycol (PEG-3). In the development of this project, a retrosynthetic analysis was first proposed for the synthesis of the chosen siderophore coupled to the linker, in which three main substructures were selected: a siderophore substructure formed by a lysine and a catechol (S-7), another siderophore substructure consisting of a glycine, a lysine and a catechol (S-12) and the linker, which contains three ethylene glycol units (C-13). Next, it was possible to synthesize and characterize each of the proposed substructures and successfully connect them to obtain the siderophore coupled with the proposed linker (SC-16). Unfortunately, the coupling tests of the polymers with the synthesized siderophore fragments were unsuccessful. In view of the results obtained and in order to be able to prepare the conjugates of interest, it is proposed to change the coupling reaction conditions and/or use other functionalized polymers. Traballo fin de grao (UDC.CIE). Química. Curso 2021/2022

Background and Objectives: Bacteriocins are considered alternative non-conventional antimicrobials produced by certain bacteria with activity against closely related species. The present study focuses on screening, characterization, and partial purification of bacteriocins produced by Staphylococcus sp. isolated from different clinical sources such as pus and blood. Materials and Methods: A total of 100 Staphylococcus isolates were screened for bacteriocin production using spot on lawn assay and agar diffusion method against five indicator bacteria. Bacteriocins from five selected highly active isolates were subjected to proteinase-K enzyme, different pH, and heating at different temperatures, and investigated the stabilities of their antimicrobials. Two selected isolates, MK65 and MK88, were molecularly identified by 16S rRNA gene sequencing, explored for the presence of 18 bacteriocin genes, and liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) was used to identify their different metabolites. Results: Twenty isolates exhibited inhibitory effect against at least one indicator bacteria. Micrococcus luteus ATCC 4698 showed the highest sensitivity to such bacteriocins. Proteinase K, acidic pH, and heating at 100°C triggered marked activity inhibition. However, amylase enzyme, alkaline pH, and heating at 80°C caused trivial effects. Four out of eighteen bacteriocin genes were detected using PCR. Fermentation, partial purification, and LC-HRESIMS of total protein extracts of two selected isolates, MK65 and MK88, revealed the production of different antimicrobial peptides. Conclusion: To the best of our knowledge, this is the first study to report the production of micrococcin and α-circulocin from Staphylococcus aureus MK65 and the production of amonabactin from Staphylococcus epidermidis MK88.

Background and Objectives: Bacteriocins are considered alternative non-conventional antimicrobials produced by certain bacteria with activity against closely related species. The present study focuses on screening, characterization, and partial purification of bacteriocins produced by Staphylococcus sp. isolated from different clinical sources such as pus and blood. Materials and Methods: A total of 100 Staphylococcus isolates were screened for bacteriocin production using spot on lawn assay and agar diffusion method against five indicator bacteria. Bacteriocins from five selected highly active isolates were subjected to proteinase-K enzyme, different pH, and heating at different temperatures, and investigated the stabilities of their antimicrobials. Two selected isolates, MK65 and MK88, were molecularly identified by 16S rRNA gene sequencing, explored for the presence of 18 bacteriocin genes, and liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) was used to identify their different metabolites. Results: Twenty isolates exhibited inhibitory effect against at least one indicator bacteria. Micrococcus luteus ATCC 4698 showed the highest sensitivity to such bacteriocins. Proteinase K, acidic pH, and heating at 100°C triggered marked activity inhibition. However, amylase enzyme, alkaline pH, and heating at 80°C caused trivial effects. Four out of eighteen bacteriocin genes were detected using PCR. Fermentation, partial purification, and LC-HRESIMS of total protein extracts of two selected isolates, MK65 and MK88, revealed the production of different antimicrobial peptides. Conclusion: To the best of our knowledge, this is the first study to report the production of micrococcin and α-circulocin from Staphylococcus aureus MK65 and the production of amonabactin from Staphylococcus epidermidis MK88.

[Resumen] La aparición y propagación de resistencias frente a los agentes antimicrobianos se ha visto acelerada en los últimos años, provocando una crisis sanitaria a nivel mundial y poniendo de manifiesto la necesidad urgente de desarrollar nuevos fármacos más efectivos. Una de las tendencias actuales para enfrentarse a las bacterias patógenas multirresistentes se basa en el uso de estrategias tipo “caballo de Troya”, que emplean portadores moleculares reconocidos por receptores específicos para facilitar la penetración en las células y limitar, en algunos casos, la aparición de resistencias. En el presente trabajo se llevó a cabo la evaluación biológica de un conjugado sideróforo-antibiótico con el que se pretende aprovechar el mecanismo de captación de hierro mediado por sideróforos para promover la absorción activa de un antibiótico. Para su construcción, el vector de entrada seleccionado fue un análogo de las amonabactinas, un sideróforo de la bacteria patógena Aeromonas salmonicida subsp. salmonicida, y como antibiótico se empleó ampicilina. Se determinó la concentración mínima inhibitoria tanto del conjugado sideróforo-antibiótico como del fragmento espaciador-ampcilina (sin vector de entrada), y se compararon con la ampicilina libre. Los resultados parecen indicar la presencia de un mecanismo mediado por el análogo de las amonabactinas que facilita la entrada del conjugado mejorando su actividad respecto a la esperada para su peso molecular. Además, se inhibe el crecimiento bacteriano con el fragmento espaciador-ampicilina, lo que demuestra que el espaciador no impide la unión de la ampicilina a su diana celular. [Abstract] The emergence and spread of resistance to antimicrobial agents has accelerated in recent years, causing a global health crisis and highlighting the urgent need to develop new more effective drugs. One of the current trends in dealing with multidrug-resistant pathogenic bacteria is based on the use of “Trojan horse” strategies, which employ molecular carriers recognized by specific receptors to facilitate cell penetration and limit, in some cases, the emergence of resistance. In the present work, the biological evaluation of a siderophore-antibiotic conjugate was carried out to exploit the iron uptake mechanism mediated by siderophores to promote the active absorption of an antibiotic. For its construction, the selected input vector was an analog of amonabactins, siderophores from the pathogenic bacterium Aeromonas salmonicida subsp. salmonicida, and ampicillin was used as the antibiotic. The minimum inhibitory concentration of both the siderophore-antibiotic conjugate and the linker-ampillin fragment (without input vector) were determined and compared to that of free ampicillin. The results seem to indicate the presence of a mechanism mediated by the amonabactin analog that facilitates the entry of the conjugate, improving its activity with respect to that expected for its molecular weight. In addition, bacterial growth is inhibited by the linker-ampicillin fragment, demonstrating that the linker does not prevent the binding of ampicillin to its cell target. Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2020/2021

[Resumen]: La aparición y propagación de resistencias frente a los agentes antimicrobianos se ha visto acelerada en los últimos años, provocando una crisis sanitaria a nivel mundial y poniendo de manifiesto la necesidad urgente de desarrollar fármacos nuevos más efectivos. En el presente trabajo se planteó el desarrollo sintético de un conjugado sideróforo-antibiótico siguiendo una estrategia tipo “caballo de Troya”, así como la construcción de una sonda fluorescente para estudiar los mecanismos implicados en los procesos de reconocimiento e internalización, empleando en ambos casos un espaciador de mayor longitud al utilizado en estudios previos. El vector de entrada seleccionado fue un análogo de las amonabactinas, un sideróforo de la bacteria patógena Aeromonas salmonicida subsp. salmonicida con el que se pretendía aprovechar el mecanismo de captación de hierro para promover la absorción activa del antibiótico (ampicilina) y el fluoróforo (sulforodamina B). El trabajo desarrollado permitió la obtención de un conjugado sideróforo-antibiótico, sin embargo, su caracterización evidenció la presencia de otros compuestos que indicaron la posible degradación del producto. Estos resultados muestran la necesidad de realizar nuevos ensayos que permitan obtenerlo con mayor grado de pureza. Además, se consiguió la funcionalización del espaciador con los grupos reactivos necesarios para formar la sonda fluorescente, pero el acoplamiento con la sulforodamina B no dio los resultados esperados. [Resumo]: A aparición e propagación de resistencias fronte os axentes antimicrobianos viuse acelerada nos últimos anos, provocando una crise sanitaria a nivel mundial e poñendo de manifesto a necesidade urxente de desenvolver novos fármacos mais efectivos. No presente traballo propúxose o desenvolvemento sintético dun conxugado sideróforoantibiótico seguindo unha estratexia tipo “cabalo de Troia”, así como a construción dunha sonda fluorescente para estudar os mecanismos implicados nos procesos de recoñecemento e internalización, empregando en ambos casos un espaciador de maior lonxitude ao utilizado en estudos previos. O vector de entrada seleccionado foi un análogo das amonabactinas, un sideróforo da bacteria patóxena Aeromonas salmonicida subsp. salmonicida co que se pretendía aproveitar o mecanismo de captación de ferro para promover a absorción activa do antibiótico (ampicilina) e o fluoróforo (sulforodamina B). O traballo desenvolto permitiu a obtención dun conxugado sideróforo-antibiótico, non obstante, a súa caracterización evidenciou a presencia doutros compostos que indicaron a posible degradación do produto. Estes resultados mostraron a necesidade de realizar novos ensaios que permitan a súa obtención cun maior grado de pureza. Ademais, conseguiuse a funcionalización do espazador cos grupos reactivos necesarios para formar a sonda fluorescente, pero o acoplamento coa sulforodamina B non deu os resultados esperados. [Abstract]: The emergence and spread of resistance to antimicrobial agents has accelerated in recent years, causing a global health crisis and highlighting the urgent need to develop more effective new drugs. In the present work, the synthetic development of a siderophoreantibiotic conjugate following a “Trojan Horse” strategy was proposed, as well as the construction of a fluorescent probe to study the mechanisms involved in the recognition and internalization processes, using in both cases a linker longer than that used in previous studies. The selected entry vector was an analog of amonabactins, a siderophore of the pathogenic bacterium Aeromonas salmonicida subsp. salmonicida with which it was intended to take advantage of the iron uptake mechanism to promote the active absorption of the antibiotic (ampicillin) and the fluorophore (sulforhodamine B). The work carried out allowed the obtaining of a siderophore-antibiotic conjugate, however, its characterization showed the presence of other compounds that indicated the possible degradation of the product. These results show the need for new studies to obtain it with a higher degree of purity. Furthermore, functionalization of the spacer with the necessary reactive groups to form the fluorescent probe was achieved, but coupling with sulforhodamine B did not give the expected results. Traballo fin de grao (UDC.CIE). Química. Curso 2020/2021

[Abstract] 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. This work was supported by grants RTI2018-093634-B-C21/C22 from the State Agency for Research (AEI) of Spain, cofunded by the FEDER Programme from the European Union (MCIN/AEI/10.13039/501100011033/FEDER). M.B. was supported by grant PID2019-103891RJ-100 from MCIN/AEI (Spain). Work in University of Santiago de Compostela and University of A Coruña was also supported by grants GRC2018/018 and GRC2018/039, respectively, from Xunta de Galicia and BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP). D.R-V. thanks Xunta de Galicia (Spain) for a predoctoral fellowship. J.C.-S. thanks to the FPU National Program (FPU16/02060) of the Spanish Ministry of Science, Innovation and Universities for a predoctoral fellowship Xunta de Galicia; GRC2018/018 Xunta de Galicia; GRC2018/039

[Resumen:] El hierro es un micronutriente esencial para todos los organismos vivos y está implicado en reacciones enzimáticas fundamentales. Una de las estrategias más comunes para la captación de hierro para las bacterias es la producción de sideróforos. El presente Trabajo de Fin de Grado se centra en la síntesis y caracterización de intermedios sintéticos sencillos, los cuales son clave en la preparación de los sideróforos tipo amonabactina, producidos por la bacteria patógena de peces Aeromonas salmonicida subsp. salmonicida. Se propuso la obtención de dos análogos A y B como se puede observar en el esquema 1. [Figura 1. Estructura de las amonabactinas y de los análogos A y B sintetizados.] En la primera parte de este trabajo se estudia la obtención del los intermedios de reacción necesarios para la preparación de los análogos A y B, así como de cualquier sideróforo de tipo catecol, estos son el ácido-2,3-diisopropoxibenzoico y el ácido 2,3-bisbenciloxibenzoico. En la segunda parte se prepara el análogo A, mediante el acoplamiento de derivados protegidos del ácido-2,3-diisopropoxibenzoico y un derivado de la L-lisina. En la última parte del trabajo se obtiene el análogo B mediante el acoplamiento de derivados protegidos del ácido 2,3-bisbenciloxibenzoico y el (5-aminopentil)carbamato de ter-butilo. La caracterización de los productos sintetizados se lleva a cabo mediante espectroscopia de resonancia magnética nuclear de protón, carbono y técnicas de espectrometría de masas. [Resumo:] O ferro é un micronutriente esencial para todos os organismos vivos e está implicado en reaccións encimáticas fundamentais. Unha das estratexias máis comúns para a captación de ferro para as bacterias é a produción de sideróforos. O presente Traballo de Fin de Grao céntrase na síntese e caracterización de intermedios sintéticos sinxelos os cales son clave na preparación dos sideróforos tipo amonabactina, producidos pola bacteria patógena de peixes Aeromonas salmonicida subsp. salmonicida. Propúxose a obtención de dous análogos A e B como se pode observar no esquema 1. [Figura 1. Estrutura das amonabactinas e dos análogos A e B sintetizados.] Na primeira parte deste traballo estúdase a obtención do os intermedios de reacción necesarios para a preparación dos análogos A e B, así como de calquera sideróforo de tipo catecol, estes son o ácido-2,3-diisopropoxibenzoico e o ácido 2,3-bisbenciloxibenzoico. Na segunda parte prepárase o análogo A, mediante o axuste de derivados protexidos do ácido- 2,3-diisopropoxibenzoico e o un derivado da L-lisina. Na última parte do traballo obtense o análogo B mediante o axuste de derivados protexidos do ácido 2,3-bisbenciloxibenzoico e o ter-butil(5-aminopentil)carbamato. A caracterización dos produtos sintetizados leva a cabo mediante espectroscopia de resonancia magnética nuclear de protón, carbono e técnicas de espectrometría de masas. [Abstract:] Iron is a essential element for all living organisms and is involved in fundamental enzymatic reactions. One of the most common strategies for iron acquisition for bacteria is the production of siderophores. This Final Degree Work is focused in the synthesis and characterization of simple synthetic intermediates which are essential in the preparation of the siderophore amonabactin, produced by the pathogenic bacteria of fish Aeromonas salmonicida subsp. salmonicida. It was proposed the synthesis of two analogs A and B as it showed in the diagram 1. Traballo fin de grao (UDC.CIE). Química. Curso 2016/2017

The growth-stimulating effects of catecholamine stress hormones have been demonstrated in many pathogens. However, catecholamine-induced growth and its underlying mechanisms remain poorly understood in Aeromonas hydrophila. The present study sought to demonstrate that norepinephrine (NE), epinephrine (Epi), dopamine (Dopa), and L-dopa stimulate the growth of A. hydrophila in iron-restricted media containing serum. NE exhibited the strongest growth stimulation, which could be blocked by adrenergic antagonists. Furthermore, it was demonstrated that NE could sequester iron from transferrin, thereby providing a more accessible iron source for utilization by A. hydrophila. The deletion of the amoA gene associated with amonabactin synthesis revealed that the amonabactin siderophore is not required for NE-stimulated growth. However, the deletion of the TonB2 energy transduction system resulted in the loss of growth promotion by NE, indicating that a specific TonB-dependent outer membrane receptor might be involved in the transport of iron from transferrin. Collectively, our data show that catecholamine sensing promotes the growth of A. hydrophila in a manner that is dependent on the TonB2 energy transduction system.

Based on the exploration of data generated by genome sequencing, a bioinformatics approach has been chosen to identify the biosynthetic pathway of the siderophores produced by Aeromonas species. The amonabactins, considered as a virulence factor, represent a family of four variants of catechol peptidic siderophores containing Dhb, Lys, Gly, and an aromatic residue either Trp or Phe in a D-configuration. The synthesis operon is constituted of seven genes named amoCEBFAGH and is iron-regulated. The cluster includes genes encoding proteins involved in the synthesis and incorporation of the Dhb monomer, and genes encoding specific nonribosomal peptide synthetases, which are responsible for the building of the peptidic moiety. The amonabactin assembly line displays a still so far not described atypical mode of synthesis that is iterative, alternative, and optional. A disruption mutant in the adenylation domain of AmoG was unable to synthesize any amonabactin and to grow in iron stress conditions while a deletion of amoH resulted in the production of only two over the four forms. The amo cluster is widespread among most of the Aeromonas species, only few species produces the enterobactin siderophore.