Nuevos avances en la RMN anisotrópica y detección de productos naturales marinos bioactivos

Programa Oficial de Doutoramento en Química Ambiental e Fundamental. 5031V01[Resumen] A continuación, se presenta un breve resumen de cada capítulo: Capítulo 1: El uso de RMN anisotrópica en el análisis estructural de moléculas pequeñas quirales se ha expandido en los años recientes. En la actualidad, las Constantes Dipolares Residuales (CDR) pueden ser aplicadas como una herramienta estándar en el mejoramiento eficiente de la determinación de la configuración relativa de compuestos de pequeño tamaño mediante RMN estándar. En la presente tesis doctoral hemos desarrollado otra metodología de RMN anisotrópica basada en medidas de anisotropía de desplazamiento químico residual de protones (1H RCSA) que ha sido aplicada, primero a moléculas modelo, estricnina, estrona, retrorsina y a-santonina, y posteriormente a nuevos productos naturales: a un meroditerpeno aislado del alga parda Sargassum muticum, a las tricloromammindas A y B aisladas del hongo no comestible Tricholoma equestre y, finalmente a un briarano de origen marino aislado de la gorgonia Briareum asbestinum recolectada de la penísula de Yucatán. Toda esta nueva metodología se ha podido realizar a través de la mejora de nuevos dispositivos de compresión y extensión de geles aplicados a tubos de RMN de menos de 5 mm. Este desarrollo de este tipo de dispositivos es una herramienta adicional que puede ser utilizada en el futuro por espectroscopistas en el campo de RMN anisotrópica. Por último, también hemos introducido un nuevo gel deuterado (PMMA-d8) derivado de polimetilmetacrilato deuterado como medio alineante. Capítulo 2: La detección y cuantificación de productos naturales presentes en trazas en mezclas complejas es aún un campo de investigación activo para los químicos. Desde la introducción en el mercado del detector de masas Orbitrap® se iniciado una revolución en las técnicas analíticas de detección. En la presente Tesis Doctoral hemos aprovechado esta tecnología para la detección y, en algunos casos, el aislamiento y la cuantificación de algunos productos naturales de origen marino de interés biológico. Las distintas aproximaciones en las técnicas de LC/HRMS empleadas fueron aplicadas a: La detection y el aislamiento de la producción de sideróforos en varias bacteria marinas patógenas: tipo vancrobactina en tres especies de Vibrio, tipo piscibactina en una cepa mutante de Vibrio alginolyticus, el sideróforo producido por las bacterias Edwardsiella tarda y Photobacterium damselae subsp. damselae. Detección y cuantification de AHLs en tres especies patógenas de Vibrio. Detección de (TTX) en el pez Diodon hystrix recolectado en El Salvador.[Resumo] A continuación amósase un resumo de cada capítulo. Capítulo 1 O uso da RMN anisotrópica na análise estrutural de moléculas pequenas quiraies, expandiuse nos anos recentes. Na actualidade, as Constantes Dipolares Residuais (CDR)poden ser aplicadas como unha ferramenta estándar na millora eficiente da determinación da configuración relativa de compostos de pequeno tamano mediante RMN estándar. Na presente Tese Doutoral desenvolvemos outra metodoloxía de RMN anisotrópica baseada en medidas de anisotropía de desprazamento químico residual de protones (1H RCSA) que foi aplicada, primeiro a moléculas modelo, estricnina, estrona, retrorsina e [alfa]-santonina, e posteriormente a novos produtos naturais: a un meroditerpeno illado do alga parda Sargassum muticum, ás tricloromammindas A e B illadas do fungo non comestible Tricholoma equestre e, finalmente a un briarano de orixe mariña illada da gorgonia Briareum asbestinum colleitada da penísula de Iucatán: Toda esta nova metodoloxía púidose realizar a través da mellora de novos dispositivos de compresión e extensión de xeles aplicados a tubos de RMN de menos de 5 mm. Este desenvolvemento deste tipo de dispositivos é unha ferramenta adicional que pode ser utilizada no futuro por espectroscopistas no campo de RMN anisotrópica. Por último, tamén introducimos un novo xel deuterado (PMMA-d8) derivado de polimetilmetacrilato deuterado como medio alineante. Capítulo 2 A detección e cuantificación de produtos naturais presentes en trazas en mesturas complexas é aínda un campo de investigación activo para os químicos. Desde a introdución no mercado do detector de masas Orbitrap® #iniciar unha revolución nas técnicas analíticas de detección. Na presente Tese Doutoral aproveitamos esta tecnoloxía para a detección e, nalgúns casos, o illamento e a cuantificación dalgúns produtos naturais de orixe mariña de interese biolóxico. As distintas aproximacións nas técnicas de LC/HRMS empregadas foron aplicadas a: A detection e o illamento da produción de sideróforos en varias bacteria mariñas patógenas: tipo vancrobactina en tres especies de Vibrio, tipo piscibactina nunha cepa mutante de Vibrio alginolyticus, o sideróforo producido polas bacterias Edwardsiella tarda e Photobacterium damselae subsp. damselae. Detección e cuantification de AHLs en tres especies patóxenas de Vibrio. Detección de (TTX) no peixe Diodon hystrix colleitado no Salvador.[Abstract] A summary of each chapter shall be presented below. Chapter 1: The use of anisotropic NMR in the structural analysis of chiral small molecules has expanded in recent years. Currently, Residual Dipolar Constants (RDC) can be applied as a standard tool in the efficient improvement of the determination of the relative configuration of small-sized compounds by standard NMR. In the present Thesis dissertation, we have developed another methodology of anisotropic NMR based on measures of anisotropy of residual chemical proton displacement (1H RCSA) that has been applied, firstly to compound-models, strychnine, estrone, retrorsine and [alfa]-santonin, and subsequently to new natural products: to an isolated meroditerpene of the brown alga Sargassum muticum, the trichloromaminades A and B isolated from the non-edible fungus Tricholoma equestre and, finally to a briarane of marine origin isolated from the gorgonian Briareum asbestinum collected from the peninsula of Yucatan (México). Chapter 2: The detection and quantification of natural products present in traces in complex mixtures is still an active field of research for chemists. Since the introduction of the Orbitrap® mass detector into the market, a revolution in analytical detection techniques has begun. In this Doctoral Thesis we have taken advantage of this technology for the detection and, in some cases, the isolation and quantification of natural products of marine origin of biological interest. The different approaches in the LC / HRMS techniques used were applied to: Detection and isolation of siderophore production in several pathogenic marine bacteria: vanchrovatin type in three Vibrio species, piscibactin type in a mutant strain of Vibrio alginolyticus, the siderophore produced by the bacteria Edwardsiella tarda and Photobacterium damselae subsp. damselae Detection and quantification of AHLs in three pathogenic Vibrio species. Detection of TTX in the fish Diodon hystrix collected in El Salvador

Detección de sideróforos presentes en bacterias patógenas en peces de acuicultura : búsqueda de funcionales adecuados para la metodología DFT-NMR en moléculas sencillas con átomos de azufre

[Resumen]Las bacterias de las familias Enterobacteriaceae y Vibrionaceae son responsables de grandes pérdidas económicas en todo el mundo. En este sentido las investigaciones para encontrar un mecanismo de control de estas son pertinentes. En este trabajo de fin de grado se ha demostrado que la síntesis de sideróforos tipo vancrobactina es recurrente en la especie Vibrio. Además hemos comprobado que la movilización por conjugación de plásmidos involucrados en la biosíntesis de sideróforos entre bacterias patógenas puede proveer de nuevas funciones a la bacteria receptora. También hemos comprobado la existencia del sideróforo vibrioferrina, propuesto por previamente por análisis genómicos, en la bacteria patógena Edwardsiella tarda. Finalmente se encontró una combinación funcional y conjunto base adecuada para la determinación desplazamientos químicos de carbono-13 mediante cálculos DFT en modelos sencillos con átomos de azufre.[Abstract]Enterobacteriaceae and Vibrionaceae bacteria are responsible for considerable economic losses worldwide. In order to find a control mechanism of this fact makes any research related very relevant. In this memory we found that the synthesis of vancrobactin-type siderophores is recurrent in all Vibrio species. Also we have demonstrated that the mobilization by plasmid conjugation involved in the biosynthesis of siderophores in pathogenic bacteria may provide new functions to a recipient bacterium. Furthermore we have detected the siderophore vibrioferrina in a pathogenic bacteria Edwardsiella tarda, prevously proposed by genomic analysis. Finally we found a suitable pair functional/basis set for determine 13C chemical shifts on simple models containing sulfur atoms.[Resumo]As bacterias da familia Enterobacteriaceae e Vibrionaceae son responsables de grandes perdas económicas en todo o mundo. E por iso moi importante todas as investigacións posibles co fin de atopar os mecanismos de control destas bacterias. Iste traballo de fin de grado mostra que a síntese de sideróforos tipo vancrobactina é recorrente en especies de Vibrio. Asemade vimos de demostrar que a mobilización por conxugación dun plasmídeo responsable na biossíntese de sideróforos en bacterias patóxenas, poden proporcionar novas funcións a bacteria receptora. Tamén determinanos que o sideróforo vibrioferrina está presente na bacteria patóxenas Edwardsiella tarda, composto proposto con 13 anterioridade pola análise xenómica. Finalmente, atopamos un funcional e un conxunto base adecuado para determinar os desplazamentos químicos de carbono-13 en modelos simples con átomos de xofre.Xunta de Galicia; 110PXIB235157PRMinisterio de Ciencia e Innovación; AGL2009-12266-C02-022009-1226Traballo fin de mestrado (UDC.CIE). Ciencias, tecnoloxía e xestión ambienta

Governance Review Commission General Rationale Rev 2

Bacterial infectious diseases produced by Vibrio are the main cause of economic losses in aquaculture. During recent years it has been shown that the expression of virulence genes in some Vibrio species is controlled by a population-density dependent gene-expression mechanism known as quorum sensing (QS), which is mediated by the diffusion of signal molecules such as N-acylhomoserine lactones (AHLs). QS disruption, especially the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), is one of the novel therapeutic strategies for the treatment of bacterial infections. In this study, we present the detection of AHLs in 34 marine Vibrionaceae strains. Three aquaculture-related pathogenic Vibrio strains, V. mediterranei VibC-Oc-097, V. owensii VibC-Oc-106 and V. coralliilyticus VibC-Oc-193 were selected for further studies based on their virulence and high production of AHLs. This is the first report where the signal molecules have been characterized in these emerging marine pathogens and correlated to the expression of virulence factors. Moreover, the results of AHL inactivation in the three selected strains have been confirmed in vivo against brine shrimps (Artemia salina) and Manila clams (Venerupis philippinarum). This research contributes to the development of future therapies based on AHL disruption, the most promising alternatives for fighting infectious diseases in aquaculture.This research was supported in part by grants from the Ministerio de Economía y Competitividad (Spain) (http://www.mineco.gob. es/portal/site/mineco/idi) (AGL2012-39274-C02- 02; AGL2015-68806-R; AGL2015-63740-C2-2-R). José Carlos Reina is supported by a FPU fellowship rom the Ministerio de Educación, Cultura y Deporte (Spain) (https://www.mecd.gob.es/ portada-mecd/) (FPU15-01717) and a Initiation to Research fellowship for Master Students (2016) from the University of Granada (Spain

The Siderophore Piscibactin Is a Relevant Virulence Factor for Vibrio anguillarum Favored at Low Temperatures

Vibrio anguillarum 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 Photobacterium damselae subsp. piscicida. The chemical characterization of this siderophore confirmed that some strains of V. anguillarum produce piscibactin. An in silico analysis of the available genomes showed that this genomic island is present in many of the highly pathogenic V. anguillarum strains lacking the anguibactin system. The construction of single and double biosynthetic mutants for vanchrobactin and piscibactin allowed us to study the contribution of each siderophore to iron uptake, cell fitness, and virulence. Although both siderophores are simultaneously produced, piscibactin constitute a key virulence factor to infect fish, while vanchrobactin seems to have a secondary role in virulence. In addition, a transcriptional analysis of the gene cluster encoding piscibactin in V. anguillarum showed that synthesis of this siderophore is favored at low temperatures, being the transcriptional activity of the biosynthetic genes three-times higher at 18°C than at 25°C. We also show that iron levels and temperature contribute to balance the synthesis of both siderophoresThis work was supported by grants AGL2015-63740-C2-1-R and AGL2015-63740-C2-2-R (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union. The support of Xunta de Galicia (Spain) with grant GRC-2014/007 is also acknowledgedS

Secreted citrate serves as iron carrier for the marine pathogen photobacterium damselae subsp damselae

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 mutant did not show any growth impairment in rich or minimal media, but it accumulated a noticeable amount of citrate (around 7 mM) in the culture medium, irrespective of the iron levels. The parental strain accumulated citrate, but in an iron-regulated fashion, being citrate levels 5–6 times higher under iron restricted conditions. In addition, a null mutant deficient in citrate synthase showed an impairment for growth at high concentrations of iron chelators, and showed almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the iron-restricted culture supernatants resulted in a CAS-positive fraction with biological activity as siderophore. HPLC purification of that fraction yielded a pure compound which was identified as citrate from its MS and NMR spectral data. Although the production of another citrate-based compound with siderophore activity cannot be ruled out, our results suggest that Pdd secretes endogenous citrate and use it for iron scavenging from the cell environmentThis work was supported by grants AGL2012-39274-C02-01/02 and AGL2015-63740-C2-1/2-R (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union. The support of Xunta de Galicia (Spain) with grant GRC-2014/007 is also acknowledgedS

Relative Configuration of Micrograms of Natural Compounds Using Proton Residual Chemical Shift Anisotropy

[Abstract] 3D molecular structure determination is a challenge for organic compounds or natural products available in minute amounts. Proton/proton and proton/carbon correlations yield the constitution. J couplings and NOEs oftentimes supported by one-bond 1H,13C residual dipolar couplings (RDCs) or by 13C residual chemical shift anisotropies (RCSAs) provide the relative configuration. However, these RDCs or carbon RCSAs rely on 1% natural abundance of 13C preventing their use for compounds available only in quantities of a few 10’s of µgs. By contrast, 1H RCSAs provide similar information on spatial orientation of structural moieties within a molecule, while using the abundant 1H spin. Herein, 1H RCSAs are accurately measured using constrained aligning gels or liquid crystals and applied to the 3D structural determination of molecules with varying complexities. Even more, deuterated alignment media allow the elucidation of the relative configuration of around 35 µg of a briarane compound isolated from Briareum asbestinum.This work was supported by the Max Planck Society and grew out of a collaboration in the context of the Forschergruppe (FOR 934) continued now by the DFG (Gr1211/19–1 and Re1007/9–1)/CAPES 418729698 project. N.N. gratefully acknowledges the financial support by SERB, New Delhi for ECR Grant with File No.: ECR/2017/001811. This work was also funded by grants RTI2018-093634-B-C22 from the State Agency for Research (AEI) of Spain, both co-funded by the FEDER Programme from the European Union, BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP) and GRC2018/039 and Agrupación Estratégica CICA-INIBIC ED431E 2018/03 from Xunta de Galicia. C.J., J.R., and D.P.P. acknowledge CESGA for the computational support. J.C.F. acknowledges predoctoral research stay grant Inditex-UDC. D.P.P. received a fellowship from the program National Council of Science and Technology (CONACYT) of Mexico and the Secretariat of Research, Innovation and Higher Education (SIIES) of Yucatan (Mexico). We also thank Dr. G. Jithender Reddy for one isotropic measurement. We also thank Dr. Christian Schmidt for his cooperation in the manufacturing of micro stretching device. ANV thanks CNPq for a research fellowship and financial support M(426216/2018–0)German Research Foundation; Gr1211/19–1German Research Foundation; Re1007/9–1Brasil. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); 418729698República de la India. Science and Engineering Research Board; ECR/2017/001811Xunta de Galicia; 0474_BLUEBIOLAB_1_EXunta de Galicia; GRC2018/039Xunta de Galicia; ED431E 2018/03Brasil. Conselho Nacional de Desenvolvimento Científico e Tecnológico; M(426216/2018–0

Antimicrobial Diterpene Alkaloids from an Agelas citrina Sponge Collected in the Yucatán Peninsula

[Abstract] Three new diterpene alkaloids, (+)-8-epiagelasine T (1), (+)-10-epiagelasine B (2), and (+)-12-hydroxyagelasidine C (3), along with three known compounds, (+)-ent-agelasine F (4), (+)-agelasine B (5), and (+)-agelasidine C (6), were isolated from the sponge Agelas citrina, collected on the coasts of the Yucatán Peninsula (Mexico). Their chemical structures were elucidated by 1D and 2D NMR spectroscopy, HRESIMS techniques, and a comparison with literature data. Although the synthesis of (+)-ent-agelasine F (4) has been previously reported, this is the first time that it was isolated as a natural product. The evaluation of the antimicrobial activity against the Gram-positive pathogens Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis showed that all of them were active, with (+)-10-epiagelasine B (2) being the most active compound with an MIC in the range of 1–8 µg/mL. On the other hand, the Gram-negative pathogenes Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were also evaluated, and only (+)-agelasine B (5) showed a moderate antibacterial activity with a MIC value of 16 μg/mL.This work was supported by grants RTI2018-093634-B-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) and BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP). This work was supported by Projects PI17/01482 and PI20/01212 awarded to AB, all within in the National Plan for Scientific Research, Development and Technological Innovation 2017–2020 and funded by the ISCIII—General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe”. The work was also supported by CIBERINFEC (CIBER de Enfermedades Infecciosas). The study was also funded by project IN607D 2021/12 (GAIN-Agencia Gallega de Innovación—Consellería de Economía, Emprego e Industria) awarded to AB. The study was also funded by projects GRC2018/039 from Xunta de Galicia. Dawrin Pech-Puch received his postdoctoral fellowship from the National Council of Science and Technology (CONACYT) of Mexico. This work was supported by the Max Planck Society and the DFG (Gr1211/19-1)/CAPES 418729698 projectXunta de Galicia; IN607D 2021/12Xunta de Galicia; GRC2018/039Deutsche Forschungsgemeinschaft = German Research Foundation; Gr1211/19-1Brasil. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); 41872969

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes

Connection of isolated stereoclusters by combining 13C-RCSA, RDC, and J-based configurational analyses and structural revision of a tetraprenyltoluquinol chromane meroterpenoid from Sargassum muticum

The seaweed Sargassum muticum, collected on the southern coast of Galicia, yielded a tetraprenyltoluquinol chromane meroditerpene compound known as 1b, whose structure is revised. The relative configuration of 1b was determined by J-based configurational methodology combined with an iJ/DP4 statistical analysis and further confirmed by measuring two anisotropic properties: carbon residual chemical shift anisotropies (13C-RCSAs) and one-bond 1H-13C residual dipolar couplings (1DCH-RDCs). The absolute configuration of 1b was deduced by ECD/OR/TD-DFT methods and established as 3R,7S,11R.Agencia Estatal de Investigación (AEI) de España | Ref. RTI2018-093634-B-C22Interreg España-Portugal | Ref. 0474_BLUEBIOLAB_1_EXunta de Galicia | Ref. GRC2018/039Xunta de Galicia | Ref. ED431E 2018/03Max Planck Society | Ref. CAPES 418729698SERB, New Delhi | Ref. ECR/2017/00181

The fish pathogen Vibrio ordalii under iron deprivation produces the siderophore piscibactin

Vibrio ordaliiis the causative agent of vibriosis, mainly in salmonid fishes, and its virulencemechanisms are still not completely understood. In previous works we demonstrated thatV. ordaliipossess 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 asiderophore byV. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF)of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able toclearly demonstrate thatV. ordaliiproduces piscibactin under iron limitation. The synthesis andtransport of this siderophore is encoded by a chromosomal gene cluster homologous to another onedescribed inV. anguillarum, which also encodes the synthesis of piscibactin. Usingβ-galactosidaseassays we were able to show that two potential promoters regulated by iron control the transcriptionof this gene cluster inV. ordalii. Moreover, biosynthetic and transport proteins corresponding topiscibactin synthesis and uptake could be identified in membrane fractions ofV. ordaliicells grownunder iron limitation. The synthesis of piscibactin was previously reported in other fish pathogenslikePhotobacterium damselaesubsp.piscicidaandV. anguillarum, which highlights the importance ofthis siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animalsThis work was supported by Grants CONICYT/FONDAP/15110027 and FONDECYT Nº 1190283 from the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT, Chile), and by grants AGL2015-63740-C2-1-R and AGL2015-63740-C2-2-R from the State Agency for Research (AEI) of Spain, both co-funded by the FEDER Programme from the European Union. Grants from Xunta de Galicia (Spain) supporting work at University of Santiago de Compostela (grant GRC2018/18) and University of A Coruña (grant GRC2018/39 and CICA-INIBIC support ED431E2018/03) are also acknowledgedS