SOUSA, T. S.; PESSOA, O. D. L. Micro-organismos marinhos produtores de metabólitos secundários biologicamente ativos. 2013. 228 f. Tese (Doutorado em Química Orgânica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by José Jairo Viana de Sousa (jairo@ufc.br) on 2014-10-15T17:59:31Z No. of bitstreams: 1 2013_tese_tssousa.pdf: 22088189 bytes, checksum: f347a4a8a1de4b931bb08902d3d1e6f7 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2015-11-24T19:18:11Z (GMT) No. of bitstreams: 1 2013_tese_tssousa.pdf: 22088189 bytes, checksum: f347a4a8a1de4b931bb08902d3d1e6f7 (MD5) / Made available in DSpace on 2015-11-24T19:18:11Z (GMT). No. of bitstreams: 1 2013_tese_tssousa.pdf: 22088189 bytes, checksum: f347a4a8a1de4b931bb08902d3d1e6f7 (MD5) Previous issue date: 2013 / This work describe the chemical and biological investigation of the extracts from the marine bacterias Pseudoalteromonas sp., Micromonospora sp., Streptomyces sp. and Kocuria sp., aiming the isolation and structural elucidation of new bioactive constituents. The chemical investigation carried out with the bacteria Pseudoalteromonas sp. lead to the isolation a red pigment identified as prodigiosin and two bile acids derivatives known as deoxycholic acid and cholic acid. The prodigiosin was evaluated against four tumor cell lines showing IC50 values similar to the positive control doxorubicin. The chemical study of Micromonospora sp. Resulted in the isolation of four new anthracyclinones designed as 4,6,11-trihydroxy-9-propryltetracene-5,12-dione; 4-methoxy-9-propyltetracene-6,11-dione; 7,8,9,10 - tetrahydro-9-hydroxy-4-methoxy-9-propiltetra-cene-6,11-dione and 10β-Carbomethoxy-7,8,9,10-tetrahydro-4,6,7α,9α,11-pentahydroxy-9-propyltetracene-5,12-dione . The cytotoxic potential of these compounds were evaluated against HCT-8cell line, two of which showed moderate cytotoxicity with IC50 values of 12.74 and 6.18 M, respectively. From Streptomyces sp. strain was isolated a ditiolpyrrolidin, established as 5-oxo-6-(N-methylformamide) -4,5 - dihydro-1,2-dithiol [4,3-b] pyrrole. This secondary metabolite was tested against six tumor cell lines, shown IC50 values of 1.66, 1.05 and 1.52 mM for the metastatic prostate lines, ovarium carcinoma and glioblastoma, respectively. The study of Kocuria sp. lead to the isolation of a new peptide, which was designed as kocurin. This compound was subjected to the tested its antimicrobial assays against several pathogens bacteria and fungal including Staphylococcus aureus strains methicillin resistant (MRSA) and Staphylococcus aureus strains tiazomicin resistant. Kocurin was strongly active against MRSA MB5393 exhibiting a MIC of 0,25µg/mL, moreover showed antibacterial activity against Bacillus subtilis and Enterococcus faecium. The structures of all isolated compounds in this work were stabilized employing spectroscopic methods such as 1H and 13C NMR (1D and 2D), mass spectrometry and infrared. / Este trabalho descreve o estudo químico e biológico dos extratos das bactérias marinhas Pseudoalteromonas sp., Micromonospora sp., Streptomyces sp. e Kocuria sp., visando o isolamento e a elucidação estrutural de novos constituintes bioativos. A investigação química realizada com a bactéria Pseudoalteromonas sp. resultou no isolamento de um pigmento vermelho identificado como prodigiosina e de dois ácidos biliares conhecidos como ácido desoxicólico e ácido cólico. A prodigiosina foi testada frente a quatro linhagens de células tumorais e apresentou valores de IC50 semelhantes ao padrão positivo. O estudo químico de Micromonospora sp. resultou no isolamento de quatro novas antraciclinonas: 4,6,11-triidroxi-9-propriltetraceno-5,12-diona; 4-metoxi-9-propiltetraceno-6,11-diona; 7,8,9,10-tetraidro-9-hidroxi-4-metoxi-9-propiltetra-ceno-6,11-diona e 10β-metoxicarbonil-7,8,9,10-tetraidro-4,6,7α,9α,11–pentaidroxi–9–propil-tetraceno-5,12-diona. Esses compostos foram avaliados quanto a sua atividade anti-tumoral frente a linhagem celular HCT-8, dois dos quais mostraram citotoxidade moderada com valores de IC50 de 12,74 e 6,18 M. O estudo da bactéria Streptomyces sp. possibilitou o isolamento de uma ditiolpirrolidina cuja estrutura foi estabelecida como 5-oxo-6-(N-metilformamida)-4,5- diidro-1,2-ditiol[4,3-b]pirrol. Esse metabólito teve sua atividade citotóxica testada frente a seis linhagens celulares tumorais, mostrando forte atividade com IC50 de 1,66, 1,05 e 1,52 µM para as linhagens de próstata metastática, carcinoma de ovário e glioblastoma, respectivamente. O estudo de Kocuria sp. resultou no isolamento de um novo peptídeo denominado como kocurina. Esse composto teve sua atividade antimicrobiana testada frente a várias bactérias e fungos patogênicos, incluindo cepas de Staphylococcus aureus resistentes a meticilina (MRSA) e cepas de Staphylococcus aureus resistentes a tiazomicina. Kocurina inibiu fortemente o crescimento de MRSA MB5393 com valores de CIM de 0,25µg/mL, além de exibir atividade antibacteriana contra as bactérias Bacillus subtilis e Enterococcus faecium. As estruturas de todos os compostos isolados neste trabalho foram determinadas empregando métodos espectroscópicos tais como RMN 1H e 13C (1D e 2D), espectrometria de massas e infravermelho.

The eukaryote-associated Pseudoalteromonas tunicata produces two pigments and several other bioactive compounds that are able to inhibit a range of marine organisms including bacteria, protozoa, fungi, algal spores and invertebrate larvae. Early studies suggested that the production of bioactive compounds is correlated with pigmentation in P. tunicata. In one of these studies, a transposon mutagenesis library identified a white mutant, wmpD-, which had been disrupted in a gene encoding a component of the type 11 secretion (T2S) machinery. The T2S system is involved in the transport of different extracellular enzymes in many bacteria. In some cases, the T2S pathway also exports proteins that remain attached to the cells. This thesis aimed to investigate the role of the T2S pathway in the production of the pigments and bioactive compounds in P. tunicata. In order to gain insight into this relationship, two proteomics approaches (2D-PAGE and iTRAQ) were applied to investigate the profile of the secreted proteins (or secretome) in P. tunicata wild-type and the white mutant wmpD-. Proteomic analysis using 2D-PAGE revealed that 23 proteins were differentially expressed between P. tunicata Wt and the mutant wmpD-. The identities of some of these proteins could be correlated with the function of the T2S system in P. tunicata. The role of one of the proteins identified using 2D-PAGE was further investigated through the construction of a gene knockout mutant (hiik mutant). The supernatant activity of the hiik mutant was compared to that of P. tunicata Wt, and it was found that the HiiA protease is required to block the activity of antimicrobial peptides, such as cecropins, produced by eukaryotic hosts in the environment. The second proteomics approach (iTRAQ) used in this thesis, enabled the relative quantitation of a number of proteins in the supernatant of P. tunicata Wt and the white mutant wmpD-. Some proteins with no function to date (hypothetical) were absent in the extracellular fraction of the wmpD- mutant, indicating they may be transported to the extracellular environment via the T2S pathway in P. tunicata. The comparative analysis of the secretome also revealed that TonS-related proteins, involved in iron acquisition, were up-regulated in the wmpD- mutant, possibly to compensate for the lack of TonS-dependant receptors in the outer membrane. Assays for iron binding activity showed that P. tunicata Wt seems to release iron binding compounds (or siderophores) constitutively into the supernatant, in contrast to the white mutant wmpD-, which responds to iron limitation by increasing the production of siderophores. Further outer membrane fractionation studies, indicated that the P. tunicata T2S system is likely to be involved in the transport of TonB-dependant receptors to the outer membrane. The overall results discussed in this thesis indicate that the T2S system has an essential role in the general physiology of P. tunicata, as for iron metabolism, as well as in the in the relationship between this bacterium and eukaryotic hosts in the environment.

No description available.

Le contrôle du biofouling sur des surfaces inertes immergées ou en atmosphère humide est une nécessité dans le secteur marin, tant pour des raisons économiques qu’environnementales. La formation de biofilm microbien, étape préalable à la formation du biofouling, est souvent intrinsèquement liée chez les bactéries au système de communication “Quorum Sensing” (QS). Chez certaines bactéries Gram négatif, le QS est basé sur la perception de petites molécules diffusibles appelées N- Acyl Homosérine Lactones (AHLs). L’une des stratégies antifouling en voie de développement de nos jours repose sur l’inhibition du QS bactérien. L’objectif de cette thèse est d’utiliser certaines bactéries marines afin d’identifier des molécules anti-QS capables de perturber la formation de biofilm. Ce travail a donc porté sur la mise en évidence de molécules AHLs impliquées dans le QS chez certaines bactéries marines isolées de la rade de Toulon, l’étude de la modulation de certains phénotypes dont la formation du biofilm, par ces molécules et, la mise en place d’un test préliminaire d’inhibition du QS. Parmi les trois bactéries isolées de la rade de Toulon (TC8, TC14 et TC15) du genre Pseudoalteromonas, connues pour produire de nombreuses molécules actives, et testées pour leur capacité à sécréter des AHLs, seule Pseudoalteromonas sp. TC15 a produit la C12-HSL. P. ulvae TC14, capable de produire un biofilm conséquent et de la violacéine, ne produit aucune AHL. Afin d’évaluer la possibilité d’utiliser une bactérie marine comme outil de criblage anti-QS, interférant avec les AHLs et les conséquences sur son biofilm, des AHLs exogènes ont été testées sur la production de violacéine, la formation de biofilm et la mobilité de TC14. Certaines AHLs ont montré qu’elles pouvaient réguler la production de violacéine et la formation de biofilm chez TC14, suggérant l’existence d’un récepteur AHLs fonctionnel. Des tests préliminaires d’inhibition du QS ont été effectués avec des molécules commerciales et des analogues synthétiques. La 3-oxo-C6-HSL commerciale, ainsi que l’esculétine et la p- benzoquinone, connues pour interférer avec le QS bactérien, ont été capables d’inhiber la production de violacéine ainsi que la formation de biofilm de TC14 à des concentrations n’affectant pas sa croissance. Cette étude suggère donc que P. ulvae TC14 pourrait être utilisée comme un outil de recherche de molécules anti-QS en conditions proches de celles trouvées dans l’environnement marin, et ce dans le but d’être ultérieurement testées sur la formation de biofilm. L’objectif à plus long terme reste de trouver un moyen de limiter la formation du biofilm en utilisant des molécules non toxiques pour l’environnement. / The biofouling control on immersed inert surfaces or in moist atmosphere is a necessity in the marine sector for both economic and environmental reasons. Microbial biofilm formation, the initial step of biofouling development, is intrinsically linked to the communication system “Quorum sensing” (QS). In some Gram negative bacteria, QS is based on the perception of small diffusible signaling molecules called Acyl Homoserine Lactones (AHLs). The inhibition of bacterial QS is part of the different antifouling strategies currently developed. This present work focused on the detection of AHLs molecules involved in this communication system in bacteria isolated from Toulon harbor and the study of modulation of some phenotypes, including biofilm formation, by these molecules as well as the development of a preliminary anti-QS assay. Three marine bacteria isolated from Toulon harbor (TC8, TC14 and TC15), belonging to the Pseudoalteromonas genus, known to synthesize many active molecules, have been tested for their ability to produce AHLs. Only Pseudoalteromonas sp. TC15 produced the C12-HSL. P. ulvae TC14 a violacein-producing and biofilm-forming bacteria, did not secrete any AHLs. Few marine bacteria are used as an anti-QS screening tool, especially by interfering with AHLs with the goal of studying the consequences on biofilm formation. In order to evaluate the possibility to use TC14 with this purpose, exogenous AHLs were tested on the violacein production, the biofilm formation and the motility of TC14. Some AHLs were able to regulate violacein production and biofilm formation suggesting the presence of a functional AHLs receptor in TC14. Preliminary QS inhibition assays were performed with commercial molecules and synthetic analogues. The commercial 3-oxo-C6-HSL as well as esculetin and p-benzoquinone, known to interfere with bacterial QS, were able to inhibit QS and biofilm formation at a non-toxic concentration. Overall, this study suggests that the marine strain P. ulvae TC14 may be used as a tool for the detection of anti-QS molecules in conditions closed to the marine environment. These molecules may subsequently be tested on the biofilm formation of marine bacteria. The long term objective is to find a way to limit biofilm formation, using non-toxic molecules for the environment.

Siderophores are secreted by marine bacteria to increase Fe uptake when Fe is limiting but are not produced when sufficient Fe is present to saturate growth. These results are well established in laboratory batch cultures of a number of isolates obtained from the open sea. Little is known, however, regarding the kinetics of siderophore secretion by heterotrophic bacteria in response to transients in Fe deprivation and resupply. We examined growth, hydroxamate siderophore concentration, and electron transport chain activity (a biochemical measure of Fe nutritional state) of Pseudoalteromonas haloplanktis, a representative gamma-proteobacterium from the Fe deficient region of the subarctic Pacific Ocean. Hydroxamate concentration was roughly 5-fold higher in batch cultures grown in low than in high Fe medium. Iron injection to the low Fe cultures repressed hydroxamic acid production and increased growth and ETC activity. Steady-state hydroxamate concentration in the chemostat increased 5-fold as Fe-limited growth rate declined from 9.8 to 2.8 d -1. This increase compounded to a 2.8-fold change in hydroxamates cell-1 reflecting the greater costs of growth at low Fe. Three types of Fe perturbation were made to Fe-limited chemostat cultures: 1) A switch perturbation that decreased the dilution rate of the chemostat-by ∼3-fold caused a transient increase in cell density that subsequently declined to a new steady state level. Hydroxamate concentration increased linearly over the same time. 2) A transient addition of dissolved Fe increased the total hydroxamate concentration in the chemostat within 1-3 hours which was followed by a decrease and then subsequent increase as the cells re-entered Fe-limitation. Dilution rate affected the response. Normalized to bacteria density, hydroxamate concentration remained constant for the first 2 hours after the Fe addition and then declined and returned to pre-infusion levels. Thus, Fe addition stimulated siderophore production by increasing the density of bacteria, which continued to secrete hydroxamates at a Fe-limited rate. 3) A continuous addition of low levels of dissolved Fe increased bacteria density and siderophore concentration. The net secretion rate of siderophores was proportional to the increase in Fe supply rate to the chemostat. At high Fe concentration, hydroxamate concentration declined to undetectable levels as the bacteria became Fe-sufficient and C-limited. Siderophore secretion by Fe-limited P. haloplanktis was repressed after 2 hours (corresponding roughly to 1-2 cell generations) following Fe re-supply.

The dark-green pigmented marine bacterium Pseudoalteromonas tunicata produces several extracellular compounds against a range of common fouling organisms including bacteria, fungi, protozoa, diatoms, invertebrate larvae and algal spores. The regulator WmpR, which has N-terminal similarity to ToxR from Vibrio cholerae and CadC from Escherichia coli, controls all of the pigment and antifouling phenotypes. These compounds appear at the onset of stationary phase. The role of WmpR as a stationary phase regulator in P. tunicata was investigated in this thesis. Starvation and stress studies demonstrated that WmpR does not appear to control genes necessary for survival during carbon, phosphate or nitrogen starvation and UV/hydrogen peroxide stress. Intriguingly, phosphate starvation caused pigmentation of wmpR mutant (D2W2) logarithmic phase cells, suggesting a second regulation of the pigments (and thus antifouling compounds) that could be mediated by the PhoR/B twocomponent regulatory system. Proteomic analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) found that 11 proteins were differentially regulated by WmpR, and the identities of some of these proteins suggested a role for WmpR as a general stationary phase regulator rather than a specific starvation or stress regulator. Gene expression studies using RNA-arbitrarily primed PCR introduced a new role for WmpR as a regulator of iron acquisition; a TonB-dependant outer membrane receptor gene and a non-ribosomal peptide synthetase (NRPS) gene were up-regulated in the stationary phase Wt strain compared to the D2W2 strain. An assay for iron-binding activity supported the proposal that the NRPS may be making a siderophore. Further studies demonstrated that WmpR is required for survival under long-term low-iron conditions and that the pigments and antifouling genes are down-regulated during low-iron, while biofilm formation is up-regulated. WmpR also appears to constitutively regulate the production of iron-binding compounds, a novel regulation of iron acquisition that has not been seen in other organisms studied so far. A model is proposed that describes WmpR as responding to environmental signals, including iron, and co-ordinating the expression of a complex regulon including a number of genes involved in iron acquisition, general stationary phase physiology and bioactive secondary metabolite production.

The marine bacterium Pseudoalteromonas tunicata produces target-specific inhibitory compounds against bacteria, algae, fungi and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. This study examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water-channels. Interestingly, a repeatable pattern of cell death in the centre of microcolonies was observed. The antibacterial and autolytic protein, AlpP, produced by P. tunicata was found to be involved in this biofilm killing and a

abstract: This thesis research focuses on phylogenetic and functional studies of microbial communities in deep-sea water, an untapped reservoir of high metabolic and genetic diversity of microorganisms. The presence of photosynthetic cyanobacteria and diatoms is an interesting and unexpected discovery during a 16S ribosomal rRNA-based community structure analyses for microbial communities in the deep-sea water of the Pacific Ocean. Both RT-PCR and qRT-PCR approaches were employed to detect expression of the genes involved in photosynthesis of photoautotrophic organisms. Positive results were obtained and further proved the functional activity of these detected photosynthetic microbes in the deep-sea. Metagenomic and metatranscriptomic data was obtained, integrated, and analyzed from deep-sea microbial communities, including both prokaryotes and eukaryotes, from four different deep-sea sites ranging from the mesopelagic to the pelagic ocean. The RNA/DNA ratio was employed as an index to show the strength of metabolic activity of deep-sea microbes. These taxonomic and functional analyses of deep-sea microbial communities revealed a `defensive' life style of microbial communities living in the deep-sea water. Pseudoalteromonas sp.WG07 was subjected to transcriptomic analysis by application of RNA-Seq technology through the transcriptomic annotation using the genomes of closely related surface-water strain Pseudoalteromonas haloplanktis TAC125 and sediment strain Pseudoalteromonas sp. SM9913. The transcriptome survey and related functional analysis of WG07 revealed unique features different from TAC125 and SM9913 and provided clues as to how it adapted to its environmental niche. Also, a comparative transcriptomic analysis of WG07 revealed transcriptome changes between its exponential and stationary growing phases. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2013

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22665号 / 農博第2420号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5296(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Pseudomonas aeruginosa est un pathogène opportuniste induisant des infections chroniques chez les patients atteints de mucoviscidose. L'éradication de ces infections est presque impossible à l'âge adulte du fait de la formation de biofilms dans les poumons des patients. Les traitements antibiotiques actuels sont peu efficaces contre les biofilms car ce mode de vie assure d'une part la protection des bactéries contre les agents anti-microbiens par l'intermédiaire de la matrice extracellulaire, et favorise d'autre part l'émergence de mécanismes de résistance. Il est donc essentiel de trouver des alternatives thérapeutiques. La bactérie marine Pseudoalteromonas sp. 3J6 sécrète une molécule à activité anti-biofilm efficace contre la souche de laboratoire P. aeruginosa PA01 et les souches cliniques P. aeruginosa MUC-N1, MUC-N2 et MUC-P4. Ces souches ont été caractérisées aux niveaux de leur formation de biofilms in vitro et de leur virulence . Ceci a montré que ces souches sont très différentes les unes des autres et qu'une seule souche, telle que la souche de laboratoire PA01, ne peut pas être représentative des profils observés. Il est donc nécessaire de mener les études anti-biofilms sur plusieurs souches, telles que celles que nous avons sélectionnées. Le potentiel thérapeutique du surnageant de culture (SNa.Js} de Pseudoalteromonas sp. 3J6 et son extrait (Ea.Js} a été étudié en évaluant leur toxicité, la réponse inflammatoire, leur impact sur la production de facteurs de virulence et leur potentiel thérapeutique . SNa.Js et Ea.Js n'étaient pas toxiques vis-à-vis des modèles testés, n'induisaient pas de réponse inflammatoire dans les poumons de souris et n'augmentaient pas la production par P. aeruginosa des facteurs de virulence quantifiés. De plus, SNa.Js s'est avèré être aussi efficace que l'antibiotique ciprofloxacine pour traiter une infection à P. aeruginosa MUC-N2 in vivo sur modèle murin. Ces résultats sont encourageants quant à un potentiel thérapeutique de la molécule à activité anti-biofilm pour contribuer au traitement des infections à P. aeruginosa chez les patients atteints de mucoviscidose. / Pseudomonas aeruginosa is an opportunistic pathogen leading to chronic infections in patients suffering of cystic fibrosis. Eradication of these infections is almost impossible in adults because of biofilm formation in patient's lungs. Current antibiotics treatments are not efficient enough against biofilms because this lifestyle first protects bacteria from antimicrobial agents via the biofilm extracellular matrix and secondly promotes the emergence of antibiotic resistance mechanisms. lt is therefore essential to find therapeutic alternatives . The marine bacterium Pseudoalteromonas sp. 3J6 secretes an anti-biofilm molecule active against the laboratory P. aeruginosa PA01 strain and the P. aeruginosa clinical strains MUC-N1, MUC-N2 and MUC­ P4. These strains were characterized at the levels of in vitro biofilm formation, and of their virulence. This part of the research work highlighted that these strains are different from one to another and that a single strain, such as the laboratory strain PA01, cannot be representative of the various patterns. Anti-biofilm studies have thus to be performed on several strains, such as the ones we selected. The therapeutic potential of the culture supernatant (SNa.Js} of Pseudoalteromonas sp. 3J6 and its extract Ea.Js was studied by evaluating their toxicity, inflammatory response, impact on virulence factors production, and therapeutic efficiency. SNa.Js and Ea.Js were not taxie against the tested models; did not induce inflammatory response in mice lungs, and did not enhance virulence factor production by clinical P. aerugonisa strains. Moreover, SNa.Js was as efficient as the ciprofloxacin antibiotic to treat an in vivo infection by P. aeruginosa MUC-N2 on mice. These results are encouraging as for a therapeutic potential of the anti-biofilm molecule to contribute at the treatment of P. aeruginosa infections of cystic fibrosis patients.