Analysis of the temperature-dependent adaptation of vibrio harveyi in seawater microcosms

216 p.En este trabajo se ha estudiado la capacidad de la bacteria marina Vibrio harveyi para adaptarse a la escasez de nutrientes a diversas temperaturas que puede encontrarse de forma natural en su ecosistema. Para ello se llevaron a cabo tres experimentos similares a 4, 20 y 30ºC en agua de mar previamente esterilizada y filtrada para eliminar posibles fuentes de alimento. Para entender los mecanismos de adaptación que usa la bacteria a estas temperaturas, se procedió a analizar microscópicamente el tamaño y forma que adquiere a lo largo del tiempo, y también se extrajo el ARN para estudiar a nivel de transcriptoma la regulación que se da en cada caso particular. Encontramos que la bacteria responde de forma diferente dependiendo de la temperatura de incubación. Como era de esperar responde bien a 20ºC, ya que V. harveyi crece bien en temperaturas templadas. Sin embargo, a 4ºC se ve afectada, y a 30ºC es incapaz de hacer frente correctamente a este estrés. Interesantemente, muchos brotes se dan a elevadas temperaturas, y ya que se ha detectado un aumento significativo en genes relacionados con la virulencia, es posible que este aumento en la patogenicidad se deba a una estrategia de la bacteria para intentar sobreponerse a la adversidad

Analysis of the temperature-dependent adaptation of vibrio harveyi in seawater microcosms

216 p.En este trabajo se ha estudiado la capacidad de la bacteria marina Vibrio harveyi para adaptarse a la escasez de nutrientes a diversas temperaturas que puede encontrarse de forma natural en su ecosistema. Para ello se llevaron a cabo tres experimentos similares a 4, 20 y 30ºC en agua de mar previamente esterilizada y filtrada para eliminar posibles fuentes de alimento. Para entender los mecanismos de adaptación que usa la bacteria a estas temperaturas, se procedió a analizar microscópicamente el tamaño y forma que adquiere a lo largo del tiempo, y también se extrajo el ARN para estudiar a nivel de transcriptoma la regulación que se da en cada caso particular. Encontramos que la bacteria responde de forma diferente dependiendo de la temperatura de incubación. Como era de esperar responde bien a 20ºC, ya que V. harveyi crece bien en temperaturas templadas. Sin embargo, a 4ºC se ve afectada, y a 30ºC es incapaz de hacer frente correctamente a este estrés. Interesantemente, muchos brotes se dan a elevadas temperaturas, y ya que se ha detectado un aumento significativo en genes relacionados con la virulencia, es posible que este aumento en la patogenicidad se deba a una estrategia de la bacteria para intentar sobreponerse a la adversidad

Current Status and Future Prospects of Marine Natural Products (MNPs) as Antimicrobials

peer-reviewedThe marine environment is a rich source of chemically diverse, biologically active natural products, and serves as an invaluable resource in the ongoing search for novel antimicrobial compounds. Recent advances in extraction and isolation techniques, and in state-of-the-art technologies involved in organic synthesis and chemical structure elucidation, have accelerated the numbers of antimicrobial molecules originating from the ocean moving into clinical trials. The chemical diversity associated with these marine-derived molecules is immense, varying from simple linear peptides and fatty acids to complex alkaloids, terpenes and polyketides, etc. Such an array of structurally distinct molecules performs functionally diverse biological activities against many pathogenic bacteria and fungi, making marine-derived natural products valuable commodities, particularly in the current age of antimicrobial resistance. In this review, we have highlighted several marine-derived natural products (and their synthetic derivatives), which have gained recognition as effective antimicrobial agents over the past five years (2012–2017). These natural products have been categorized based on their chemical structures and the structure-activity mediated relationships of some of these bioactive molecules have been discussed. Finally, we have provided an insight into how genome mining efforts are likely to expedite the discovery of novel antimicrobial compounds

The P. aeruginosa effector Tse5 forms membrane pores disrupting the membrane potential of intoxicated bacteria

The type VI secretion system (T6SS) of Pseudomonas aeruginosa injects effector proteins into neighbouring competitors and host cells, providing a fitness advantage that allows this opportunistic nosocomial pathogen to persist and prevail during the onset of infections. However, despite the high clinical relevance of P. aeruginosa, the identity and mode of action of most P. aeruginosa T6SS-dependent effectors remain to be discovered. Here, we report the molecular mechanism of Tse5-CT, the toxic auto-proteolytic product of the P. aeruginosa T6SS exported effector Tse5. Our results demonstrate that Tse5-CT is a pore-forming toxin that can transport ions across the membrane, causing membrane depolarisation and bacterial death. The membrane potential regulates a wide range of essential cellular functions; therefore, membrane depolarisation is an efficient strategy to compete with other microorganisms in polymicrobial environments.We gratefully acknowledge the Laboratories of Dr. Daniel Ladant (Institut Pasteur, Paris) and Dr. Victor de Lorenzo (Centro Nacional de Biotecnologia, Madrid) for the plasmids received (pKTop and pSEVA plasmids, respectively). Also, we would like to acknowledge the Laboratory of Dr. Joseph Mougous for the P. aeruginosa strains received. The technical assistance from Cristina Civantos and Adrian Ruiz is also very much appreciated. We acknowledge the FGCZ for the mass spectrometry analyses and the technical support (Functional Genomics Center Zurich (FGCZ), University/ETH Zurich). D.A.-J. acknowledges support by the MINECO Contracts CTQ2016-76941-R and PID2021-127816NB-I00, Fundacion Biofisica Bizkaia, the Basque Excellence Research Centre (BERC) programme, and IT709-13 and IT1745-22 of the Basque Government, and Fundacion BBVA. A.G.-M. acknowledges the financial support received from the Spanish Ministry of Universities and the Grants for the requalification of the Spanish university system for 2021-2023, financed by the European Union-Next Generation EU-Margarita Salas Modality. A.A. acknowledges support from the Spanish Ministry of Science and Innovation (Project 2019-108434GB-I00 funded by MCIN/AEI/10.13039/501100011033), Generalitat Valenciana (project AICO/2020/066) and Universitat Jaume I (project UJI-B2018-53). M.Q.-M. acknowledges support from the Spanish Ministry of Science and Innovation (Project IJC2018-035283-I funded by MCIN/AEI/10.13039/501100011033) and Universitat Jaume I (project UJI-A2020-21). P.B acknowledges the financial support received from the Spanish Ministry of Science and Innovation through the Ramon y Cajal Programme (contract RYC2019-026551-I)

Analysis of laccase-like enzymes secreted by fungi isolated from a cave in northern Spain

[EN] Laccases belong to a family of multicopper enzymes able to oxidize a broad spectrum of organic compounds. Despite the well-known property of laccases to carry out bleaching and degradation of industrial dyes and polyphenolic compounds, their industrial use is often limited by the high cost, low efficiency, or instability of these enzymes. To look for new microorganisms which produce laccases that are potentially suitable for industrial applications, we have isolated several fungal strains from a cave in northern Spain. Their phenotypic analysis on agar plates supplemented with ABTS (2,2 '-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) disclosed two laccase-positive strains. Further genotyping revealed that they belonged to the Gliomastix murorum and Conidiobolus thromboides species. The secretion of G. murorum and C. thromboides laccase-like enzymes was then confirmed by zymography. Further identification of these polypeptides by mass-spectroscopy revealed the nature of the laccases and made it possible to predict their functional domains and other features. In addition, plate assays revealed that the laccases secreted by both G. murorum and C. thromboides were capable of degrading industrial dyes (Congo Red, Indigo, and Eriochrome Black T). Homology modeling and substrate docking predicted the putative structure of the currently uncrystallized G. murorum enzyme as well as its amino acid residues potentially involved in interactions with these dyes. In summary, new biochemical and structural insights into decolorization mediated by G. murorum laccase as well as identification of laccase-like oxidase in C. thromboides point to a promising future for these enzymes in biotechnology.AIOTEK, Grant/Award Number: SPE12UN84; Basque Foundation for Science; Basque Government, Grant/Award Number: PRE-2013-1-90

Addressing the Joint Impact of Temperature and pH on Vibrio harveyi Adaptation in the Time of Climate Change

Global warming and acidification of the global ocean are two important manifestations of the ongoing climate change. To characterize their joint impact on Vibrio adaptation and fitness, we analyzed the temperature-dependent adaptation of Vibrio harveyi at different pHs (7.0, 7.5, 8.0, 8.3 and 8.5) that mimic the pH of the world ocean in the past, present and future. Comparison of V. harveyi growth at 20, 25 and 30 °C show that higher temperature per se facilitates the logarithmic growth of V. harveyi in nutrient-rich environments in a pH-dependent manner. Further survival tests carried out in artificial seawater for 35 days revealed that cell culturability declined significantly upon incubation at 25 °C and 30 °C but not at 20 °C. Moreover, although acidification displayed a negative impact on cell culturability at 25 °C, it appeared to play a minor role at 30 °C, suggesting that elevated temperature, rather than pH, was the key player in the observed reduction of cell culturability. In addition, analyses of the stressed cell morphology and size distribution by epifluorescent microscopy indicates that V. harveyi likely exploits different adaptation strategies (e.g., acquisition of coccoid-like morphology) whose roles might differ depending on the temperature–pH combination.The work was supported by IKERBASQUE (Basque Foundation for Science) as well as by Basque Government Grants PIBA_2021_1_0047 and MIMAS IT1657-22

Analysis of the temperature-dependent adaptation of vibrio harveyi in seawater microcosms

216 p.En este trabajo se ha estudiado la capacidad de la bacteria marina Vibrio harveyi para adaptarse a la escasez de nutrientes a diversas temperaturas que puede encontrarse de forma natural en su ecosistema. Para ello se llevaron a cabo tres experimentos similares a 4, 20 y 30ºC en agua de mar previamente esterilizada y filtrada para eliminar posibles fuentes de alimento. Para entender los mecanismos de adaptación que usa la bacteria a estas temperaturas, se procedió a analizar microscópicamente el tamaño y forma que adquiere a lo largo del tiempo, y también se extrajo el ARN para estudiar a nivel de transcriptoma la regulación que se da en cada caso particular. Encontramos que la bacteria responde de forma diferente dependiendo de la temperatura de incubación. Como era de esperar responde bien a 20ºC, ya que V. harveyi crece bien en temperaturas templadas. Sin embargo, a 4ºC se ve afectada, y a 30ºC es incapaz de hacer frente correctamente a este estrés. Interesantemente, muchos brotes se dan a elevadas temperaturas, y ya que se ha detectado un aumento significativo en genes relacionados con la virulencia, es posible que este aumento en la patogenicidad se deba a una estrategia de la bacteria para intentar sobreponerse a la adversidad

Current Status and Future Prospects of Marine Natural Products (MNPs) as Antimicrobials

The marine environment is a rich source of chemically diverse, biologically active natural products, and serves as an invaluable resource in the ongoing search for novel antimicrobial compounds. Recent advances in extraction and isolation techniques, and in state-of-the-art technologies involved in organic synthesis and chemical structure elucidation, have accelerated the numbers of antimicrobial molecules originating from the ocean moving into clinical trials. The chemical diversity associated with these marine-derived molecules is immense, varying from simple linear peptides and fatty acids to complex alkaloids, terpenes and polyketides, etc. Such an array of structurally distinct molecules performs functionally diverse biological activities against many pathogenic bacteria and fungi, making marine-derived natural products valuable commodities, particularly in the current age of antimicrobial resistance. In this review, we have highlighted several marine-derived natural products (and their synthetic derivatives), which have gained recognition as effective antimicrobial agents over the past five years (2012–2017). These natural products have been categorized based on their chemical structures and the structure-activity mediated relationships of some of these bioactive molecules have been discussed. Finally, we have provided an insight into how genome mining efforts are likely to expedite the discovery of novel antimicrobial compounds

The P. aeruginosa type VI secretion system effector Tse5 forms ion-selective membrane pores that disrupt the membrane potential of intoxicated cells

Póster presentado al 8th International Iberian Biophysics Congress celebrado en Bilbao los días 20 y 21 de junio de 2022.Pseudomonas aeruginosa is an opportunistic pathogen of great clinical impact, being the main cause of acute and chronic infections in immunosuppressed and cystic fibrosis patients. This virulence is largely due to the secretion systems possessed by the bacteria, among which the Type 6 Secretion System (T6SS) in P. aeruginosa stands out 1. The T6SS is a nanomachine that assembles inside the bacteria and injects severa effectors/toxins into target cells 2. The large number of effectors and their functional diversity play a crucial role in the virulence of the infection caused by this gram-negative bacterium. One of these effectors is the Type VI secretion system exported effector 5 (Tse5) which was described to have bacteriolytic activity although its molecular activity is still unknown 3,4. In this work we studied the molecular function of Tse5 effector. To do so, we performed growth inhibition curves to determine if it has a bacteriolytic or bacteriostatic effect. We hypothesised that the toxic domain of Tse5 (Tse5-CT) exerts its toxic activity on the membrane since its immunity protein (Tsi5) inserts into the inner bacterial membrane to neutralise the toxicity 3. To define if this toxin can increase cell permeability or disrupt membrane potential of the target bacteria, we carried out flow cytometry experiments in Pseudomonas putida. Furthermore, we designed a Tse5-CT deletion mutants with a dual reporter localize at the C-terminus that allowed to identify transmembrane regions. Our data indicate that Tse5-CT expression has a bacteriolytic effect on Pseudomonas putida cells that can be reversed by co-expression of the cognate immunity protein Tsi5. Moreover, Tse5 toxin inserts into the membrane through transmembrane and amphipathic domains and disrupts membrane potential of target cells. The results obtained in this study help to gain insight regarding the function and the mechanisms of action Tse5 effector and eventually could help to develop drugs that mimic its behaviour