Book of Abstracts of MICROBIOTEC09

Sítio da conferência: http://www.deb.uminho.pt/microbiotec09/This book contains the abstracts presented at the 3rd joint meeting of the Portuguese Society of Microbiology and The Portuguese Society of Biotechnology - MicroBiotec09, held in Vilamoura, Portugal, over 3 days, from the 28th to the 30th of November, 2009. MicroBiotec09 comes in the sequence of previous conferences organized by each society, since 1982, date of the I Encontro Nacional de Biotecnologia (Lisbon), till 2005, date of the first joint meeting - MICRO'05 + BIOTEC'05 (Póvoa de Varzim). Following this joint meeting, another - MICRO 07 + BIOTEC 07 + XXIII JPG took place in Lisbon (2007). MicroBiotec09 is a joint organization of “Sociedade Portuguesa de Biotecnologia”, “Sociedade Portuguesa de Microbiologia”, Institute for Biotechnology and Bioengineering (Universidade do Minho – Departamento de Engenharia Biológica) and Centro de Recursos Microbiológicos (Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia – Departamento de Ciências da Vida). MicroBiotec09 brings together both young and established researchers and end users to discuss recent developments in different areas of Biotechnology and Microbiology. The conference program has thus been divided in 8 major sessions: Microbial Physiology, Molecular Biology and Functional Genomics; Cell and Tissue Engineering, Biomaterials and Nanobiotechnologies; Clinical Microbiology and Epidemiology; Environmental Microbiology and Biotechnology; Health and Pharmaceutical Biotechnology; Cellular Microbiology and Pathogenesis; Industrial and Food Microbiology and Biotechnology; Bioinformatics, Comparative Genomics and Evolution. A special session to celebrate the 200th anniversary of Charles Darwin's birth and the 150th anniversary of the publication of his landmark work “On the Origin of Species by Means of Natural Selection” will also take place. A total of 295 abstracts are included in the book, consisting of 6 invited lecturers, 10 oral presentations and 44 short oral presentations given in 3 parallel sessions, along with 4 slots for viewing poster presentations. All abstracts have been reviewed and we are grateful to the members of scientific and organizing committees for their evaluations. It was an intensive task since 328 submitted abstracts were received. It has been an honor for us to contribute to setting up MicroBiotec09 during an intensive period of 6 months. We wish to thank the authors who have contributed to yield a high scientific standard to the program. We are thankful to the sponsors who have contributed decisively to this event. We also extend our gratefulness to all those who, through their dedicated efforts, have assisted us in this task. On behalf of the Scientific and Organizing Committees we wish you that together with an interesting reading, the scientific program and the social moments organized will be memorable for all.Fundação para a Ciência e a Tecnologia (FCT

Covalent modification of antimicrobial and anticancer peptides for the improvement of their farmacological properties

Following the information obtained by a rational design study, cyclic and dimeric helical stabilized analogues of the peptide Cm-p5 were synthetized. The cyclic monomer showed an increased activity in vitro against Candida albicans and Candida parapsilosis, compared to Cm-p5. Initially, fourteen mutants of Cm-p5 were synthesized following a rational design to improve the antifungal activity and pharmacological properties. Antimicrobial testing showed that the activity was lost in every of these fourteen analogues, suggesting as a main conclusion, that a Glu-His salt bridge could stabilize Cm-p5 helical conformation during the interaction with the plasma membrane. A derivative, obtained by substitution of Glu and His for Cys, was synthesized and oxidized with the generation of a cyclic monomer with improved antifungal activity. In addition, two dimers were generated during the oxidation procedure, a parallel and anti-parallel one. The dimers showed a helical secondary structure in water, whereas the cyclic monomer only showed this conformation in SDS. In addition, the antiparallel dimer showed a moderate activity against Pseudomonas aeruginosa and a significant activity against Listeria monocytogenes. Nor the cyclic monomer nor the dimers were toxi c against macrophages or THP-1 human cells. Continuing with the covalent modifications of the Cm-p5 structure, in chapter 2 is described the design, synthesis and characterization of 15 lipidated and cyclo-lipidated analogues of Cmp5. Previous studies showed that N-lipidation of Cm-p5 by Ugi-4CR increase notably the antifungal activity. Our initial biological test showed that lipidation with decanoic acid did not display any effect inthe activity of Cm-p5, while pentadecanoic acid decreased it. These results are not in accord with the increased activity in case of the Ugi lipidated analogue. The Ugi-4CRdo not only introduces a lipid chain, also produce N-substitution that can alter the conformational behavior of the lipid chain. To determine the influence of N-substitution, three analogues containing Ala, Gly or Pro between the lipid chain and the normal sequence of Cmp5 was synthetized. Gly and Pro are implied in several conformational changes in proteins or peptides.Gly possesses high flexibility while Pro is the only N-alkylated aminoacid and participate in loop or rigid structure formation. In addition, following the expected increase of activity by disulfide bridge formation, similar variants possessing Ala (introduced as a comparison), Gly or Pro between adodecyl chain and cyclic/dimeric CysCysCm-p5 were XVII prepared. Finally, lipidationby Ugi-4CR with n-dodecylisonitrile and subsequent cyclization between Cys produce cyclic and dimeric versions of Ugi lipidated cyclopeptides. All compoundsare under biological evaluation thatwill permit to gainconclusion about the structural motif needed to produce the more antifungal cyclic and lipidated analogue of Cm-p5. In chapter 3, we used the combination of solid phase and liquid phase methods for the synthesis of the anticancer drug, carfilzomib (CFZ). The convergent route comprises the tetrapeptide construction in solid phase followed by coupling with the previously prepared Leu-epoxide. Side effects of CFZ include the heart failure and shortness of breath so that the development of bioconjugation for targeted delivery is desired. With this aim, we prepared a traceless carbonylacrylic-Val-Cit-PAB linker as an alcohol in solid phase using the special DHP resin in high yield. This alcohol linker was tosylated and iodinated with the aim of improve the yields of the substitution reaction with CFZ (substitution of tosylate need heating). The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. We pretend to merge the capacity of tumor cell selectivity of pHLIP peptide with the traceless, stability and selective delivery of the new carbonylacrylic linker. Finally, the bioconjugation of CFZ to pHLIP peptide through the carbonylacrylic-Val-Cit-PAB linker, for the traceless release and targeted delivery to tumors could significantly improve the effectiveness of this drug in cancer treatment.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Seguindo a informação obtida por um estudo de concepção racional, sintetizaram-se análogos estabilizados helicoidais cíclicos, diméricos e lipidados do péptido Cm-p5. O monômero cíclico apresentou atividade aumentada in vitro contra Candida albicans e Candida parapsilosis, em comparação com Cm-p5. Inicialmente, 14 mutantes de Cm-p5 foram sintetizados seguindo um racional de sinal para melhorar a atividade antifúngica e as propriedades farmacológicas. Testes antimicrobianos mostraram que a atividade foi perdida em cada um dos 14 análogos, sugerindo como uma conclusão principal, que uma ponte de sal Glu-His poderia estabilizar a conformação helicoidal de Cm-p5 durante a interação com a membrana plasmática. Um derivado, obtido por substituição de Glu e His por Cys, foi sintetizado e oxidado com a geração de um monômero cíclico com atividade antifungica melhorada. Além disso, dois dímeros foram gerados durante o procedimento de oxidação. Os dímeros mostraram uma estrutura secundária helicoidal em água, enquanto o monômero cíclico mostrou apenas esta conformação em SDS. Além disso, o dímero antiparalelo apresentou moderada atividade contra Pseudomonas aeruginosa e atividade significativa contra Listeria monocytogenes. Nem o monómero cíclico nem os dímeros eram tóxicos contra macrófagos ou células humanas THP-1. Continuando com as modificações covalentes da estrutura do Cm-p5, no capítulo 2 se descreve o desenho, síntese e caracterização de 15 análogos lipídicos e cíclicos/lipidados doCm-p5. Estudos anteriores mostram que a N-lipidação do Cm-p5 pela reação de Ugi-4C aumenta notavelmente a atividade antifúngica. Nosso teste biológico inicial mostra que a lipidação com ácido decanóico não mostrou nenhum efeito na atividade do Cm-p5, enquanto o ácido pentadecanóico o diminuiu. Esse resultado não está de acordo com o aumento da atividade no caso do análogo lipidado da reação de Ugi-4C. A reação de Ugi-4C não apenas introduz uma cadeia lipídica, também produz uma N-substituição que pode alterar o comportamento conformacional da cadeia lipídica. Para determinar a influência da N-substituição, três análogos contendo Ala, Gly ou Pro entre a cadeia lipídica e a sequência normal de Cm-p5 foram sintetizados. Gly e Pro estão implicados em várias mudanças conformacionais em proteínas ou peptídeos. Gly possui alta flexibilidade enquanto a Pro é o único aminoácido N-alquilado e participa na formação de giros ou estruturas rígidas. Adicionalmente, seguindo o esperado aumento de actividade por formação de ponte disulfeto, foram preparadas variantes XV semelhantes possuindo Ala (introduzida como comparação), Gly ou Pro entre a cadeia de decanoilo e o peptídeo CysCysCm-p5 cíclico/dimérico. Finalmente, a lipidação por reação Ugi-4C com n-dodecilisonitrila e posterior ciclização entre Cys produzem versões cíclicas e diméricas de ciclopeptídeos Ugi lipidados. Todos os compostos estão sob avaliação biológica que permitirá obter uma conclusão sobre o motivo estrutural necessário para produzir o análogo cíclico e lipidado do Cm-p5 mais antifúngico. No capítulo 3, usamos a combinação de métodos de fase sólida e fase líquida para a síntese da droga anticâncer, carfilzomib (CFZ). A via convergente compreende a construção de tetrapéptidos em fase sólida seguida de acoplamento do epóxido preparado previamente. Os efeitos colaterais da CFZ incluem a insuficiência cardíaca e a falta de ar, de modo que o desenvolvimento da bioconjugação ou da entrega direcionada é realmente desejado. Com este objetivo, nós preparamos um ligante que não deixa resíduos, carbonilacrilic-Val-Cit-PABna forma de álcool em fase sólida usando a resina especial DHP em alto rendimento. Este ligante na forma de álcool foi tosilado e iodado com o objetivo de melhorar os rendimentos da reação de substituição com CFZ. Os peptídeos de inserção a pH baixo (pHLIPs) visam a acidez nas superfícies das célulascancerígenas e apresentam utilidade numa vasta gama de aplicações, incluindo imagiologia de tumores e distribuição intracelular de agentes terapêuticos. Neste trabalho, pretendemos fundir a capacidade de seletividade de células tumorais do peptídeo pHLIP com a libertação sem resíduos, estabilidade e seletividade do novo ligante carbonilacrílico. Finalmente, a bioconjugação de CFZ com o peptídeo pHLIP através do ligante carbonilacrílico-Val-Cit-PAB, para a libertação sem resíduos e distribuição direcionada para tumores poderia melhorar significativamente a efetividade deste fármaco no tratamento do câncer.CAPES: código de financiamento - 00

Computational systems biology methods for functional classification of membrane proteins and modeling of quorum sensing in Pseudomonas aeruginosa

Due to the function of membrane proteins and the effort required for experimental annotations, bioinformatical approaches to functionally classify uncharacterized sequences are desirable. For this, the similarity between sequences of different membrane proteins was statistically analyzed based on several amino acid compositions. To discriminate between functional classes, a ranking method was developed. We showed that including further information in the amino acid composition and filtering into different sequence regions improved the classification quality. Subsets based on function achieved sensitivities of about 80%, whereas those of random subsets are in the range of 30--35%. The pathogen Pseudomonas aeruginosa produces many virulence factors that are regulated by Quorum sensing. The number of infecting strains with antibiotic resistance is growing. Thus, new strategies focus on Quorum sensing inhibitors that target the regulatory pathways of virulence factors. Pseudomonas aeruginosa contains three Quorum sensing systems that were simulated with an extended multi--level logical formalism to study the influence of Quorum sensing inhibitors on the autoinducer and virulence factor formation. A topology analysis suggested that the proteins PqsR and PqsE act as receptors. Both are required together with an autoinducer to form pyocyanin. Enzyme inhibitors were more useful to block the autoinducer formation, whereas PqsR antagonists inhibited the pyocyanin biosynthesis stronger.Aufgrund der Funktionen von Membranproteinen und dem Aufwand experimenteller Charakterisierungen sind bioinformatische Ansätze zur Klassifizierung unbekannter Sequenzen sinnvoll. Daher wurde deren Ähnlichkeit basierend auf verschiedenen Aminosäurenkompositionen bestimmt und statistisch analysiert. Eine Ranking--Methode wurde zur Einteilung in funktionelle Klassen entwickelt. Wir konnten zeigen, dass die Vorhersagegenauigkeit durch Hinzunahme weiterer Informationen und durch Unterscheidung verschiedener Sequenzregionen verbessert werden kann. Proteingruppen mit derselben Funktion erzielten Sensitivitäten von etwa 80%, während zufällig zusammengestellte Gruppen nur 30--35% erreichten. Der Krankheitserreger Pseudomonas aeruginosa produziert viele durch Quorum Sensing regulierte Virulenzfaktoren. Wegen der wachsenden Anzahl Antibiotika--resistenter Stämme greifen neue antibakterielle Strategien gezielt diese Regulationsmechanismen an. Die drei Quorum Sensing--Systeme von Pseudomonas aeruginosa wurden mit einem erweiterten logischen Formalismus modelliert um den Einfluss von Quorum Sensing--Inhibitoren auf die Bildung von Autoinducern und Virulenzfaktoren zu untersuchen. Eine Topologie--Analyse zeigte, dass die Proteine PqsR und PqsE anscheinend als Rezeptoren zusammen mit einem Autoinducer Pyocyanin regulieren. Enzym--Hemmstoffe waren besser geeignet, die Bildung von Autoinducern zu blockieren, während PqsR--Antagonisten die Pyocyanin--Biosynthese besser hemmten

Quorum sensing dynamics in the alpha-proteobacterium Sinorhizobium meliloti at the single-cell and population level

In quorum sensing, bacteria produce and release so-called autoinducers that accumulate in the environment while the cells grow. Once these molecules reach a threshold concentration, they trigger major behavioral changes in the population. Since the triggered behaviors are thought to be effective only when performed by a large enough group, autoinducers are generally taken to indicate when this sufficient cell density has been reached. However, little is known about how these components interact dynamically at the single-cell level to fulfill their task of cell-cell communication. Furthermore, quorum sensing is often studied in well-shaken liquid cultures, but little is known about autoinducer dispersal and response dynamics over larger distances in physiological niches like the rhizosphere where active mixing is negligible. The aim of this work therefore was to investigate these aspects in the model organism Sinorhizobium meliloti. In (Bettenworth et al., 2022.), quorum sensing dynamics were investigated with respect to autoinducer synthase gene expression in single cells and the timing of the response in the respective colonies. Surprisingly, in S. meliloti the autoinducer synthase gene is not expressed continuously, but in discrete stochastic pulses. Stochasticity stems from scarcity and, presumably, low binding affinity of the essential transcription activator. Physiological factors modulate abundance of this activator or its binding affinity to the autoinducer synthase gene promoter and thereby modulate gene expression pulse frequency. Higher or lower pulse frequencies in turn trigger the onset of the quorum sensing response at lower or higher cell numbers, respectively. In other words: S. meliloti quorum sensing is based on a stochastic regulatory system that encodes each cell’s physiological condition in the pulse frequency with which it expresses its autoinducer synthase gene; pulse frequencies of all members of a population are then integrated in the common pool of autoinducers. Only once this vote crosses the threshold, the response behavior is initiated. Consequently, S. meliloti quorum sensing is not so much a matter of counting cell numbers as suggested by the analogy of the quorum, but more comparable to a voting in a local community, or the collective decision-making described for social insects (Bettenworth et al., 2022). In (Bettenworth et al., 2018), the dynamics of autoinducer dispersal by diffusion in a two-dimensional environment were explored. At first sight, diffusive spreading should yield a dilution of the molecules and, with increasing distance from the source, slow down progression of the concentration level necessary to trigger a response in distantly located receiver cells. In contrast to this expectation, however, this threshold concentration did not decelerate in respective experiments, but instead travelled with constant speed, comparable to front propagation in pattern-forming systems. According to a mathematical model, this effect was due to the exponential growth of the sender cells which yielded adding-up of an exponentially growing number of autoinducer concentration profiles, thus compensating for the thinning effect of diffusion. Consequently, even a single sender colony could induce a response in receiver cells up to 7 mm away (Bettenworth et al., 2018)

Structural and Functional Studies of Polyketide Synthases

Polyketides, natural products produced by multi-domain polyketide synthases (PKSs), have proven to be excellent starting points for drug discovery. Rational engineering of PKSs holds much promise for the generation of novel polyketide pharmaceuticals, however to enable this we need to gain a better understanding of how the mature polyketides are generated and how individual modules within a polyketide synthase assemble and interact. Here, work was performed to investigate three polypeptides from natural product indanomycin and rhizoxin biosynthesis, including the candidate polyketide cyclase IdmH, the fourth subunit of the indanomycin megasynthase, IdmO, and the branching module from the rhizoxin PKS. Indanomycin needs to undergo several transformations by post-PKS tailoring enzymes. One such enzyme, IdmH, has been hypothesised to act as a cyclase and catalyse the formation of the indane ring via a Diels-Alder reaction. Crystal structure of the wild-type IdmH was determined to 2.7 Å resolution and the interactions between IdmH and its proposed product indanomycin were characterised using NMR spectroscopy and in silico methods. Fully-reducing IdmO module was successfully expressed and purified. Characterisation by negative-stain electron microscopy resulted in a low-resolution model of IdmO, while attempts to carry out cryo-electron microscopy (cryo-EM) analysis revealed a number of difficulties associated with the denaturation of this large complex during cryo-EM grid preparation. A similar cryo-EM approach was utilised to study the branching module from the rhizoxin PKS. A 3.7 Å resolution map was determined for this module containing the ketosynthase, branching and acyl carrier protein (ACP) domains. Two ACP binding sites were identified, which can help explain the unorthodox activity of this module. This research has provided valuable insights into different aspects of PKS biology ranging from polyketide tailoring and branching to the assembly of the intact modules and forms a solid basis for future studies of these fascinating biosynthetic machines