Trichomonas vaginalis is a human pathogen causing trichomoniasis, one of the most common non-viral sexually transmitted diseases in both men and women. Trichomoniasis is currently treated with metronidazole, but the pathogen is known to develop resistance against this drug. However as the pathogen is eukaryotic, the targets for the pathogen elimination without seriously affecting the host are limited. Throughout the evolution Trichomonas vaginalis adapted to anaerobic environments by developing an alternative metabolism resulting in a reduced form of mitochondria named hydrogenosome. Hydrogenosomes lack genetic information, therefore all its proteins are nucleus-encoded and need to be transported inside the hydrogenosome using a targeting N-terminal presequence. The peptidase recognizing and cleaving those presequences at the entrance of the organelle, the hydrogenosomal processing peptidase (HPP), is unique for hydrogenosomes and therefore represents a potential drug target against the pathogen. In this work the HPP's substrate specificity towards the targeting presequences was investigated. To do so a proteomic analysis of the proteome of Trichomonas vaginalis hydrogenosomes was performed using a novel optimized protocol for N-terminal peptide sequencing. N-terminal peptides were captured using a...

Made available in DSpace on 2016-06-02T19:02:44Z (GMT). No. of bitstreams: 1 6412.pdf: 14036212 bytes, checksum: dc6e90045bca1050bc9fd76f94f1f30a (MD5) Previous issue date: 2014-06-30 / Financiadora de Estudos e Projetos / The increase in the production of antimicrobial metabolites by Paenibacillus polymyxa RNC-D was appraised through the study of cultivation variables. Two process variables, namely the glucose and inoculum concentrations, were evaluated in different levels (5 to 40 g/l, and 2.5% to 5.0% v/v, respectively), and their effects on biomass formation, minimal inhibitory concentration (MIC) against Escherichia coli and surface tension reduction (STR) were studied. The fermentation process was firstly carried out using non-optimized parameters, where the dependent variables biomass, MIC and STR reached the values of 0.6 g/l, 1.000,0 μg/ml and 18.4 mN/m, respectively. The optimum glucose (16 g/l) and inoculum concentrations (5.0% v/v) were defined in order to maximize the biomass formation, with low value of MIC and large STR of extract. Under these conditions, a biomass of 2.76 g/l, MIC of 15.8 μg/ml, and STR of 14.58 mN/m were predicted by the model. Data attained by experiments using optimized settings showed the following values: biomass 2.05 g/l; MIC 31.2 μg/ml; STR 10.7 mN/m. Thus, the percentage of improvement for each target response was: biomass 241.6%; MIC 96.88%; STR 41.85%. It was found that high concentrations of glucose substrate, although reflected in an increase in bacterial biomass, inhibited the microbial secondary metabolism, resulting in a low production of biomolecules associated with high values of MICs. Thus, initial concentrations of glucose and inoculum are shown as variables of strong influence in the production of antimicrobial metabolites by P. polymyxa RNC-D. Through the methods of experimental factorial design and surfaceresponse followed by graphical optimization it was possible to determine the optimum operating condition to achieve both maximum biomass and RTS as well as and lowest possible values of CIM. The validity of the proposed model was verified and confirmed. This is the first study on the optimization of culture conditions for the production of antimicrobial metabolites by P. polymyxa RNC-D, and constitutes an important step in the development of strategies to modulate the production of antimicrobial molecules by this microorganism in elevated levels. Novel antimicrobial compounds were isolated from the fermentation broth of P. polymyxa RNC-D, here named total extract (TE). It was possible to verify the presence of lipopeptide and peptide active compounds through enzymatic assays made with ET. Total extract was subjected to a two-phase system, resulting in lipopeptide extract (LPE) and aqueous fraction (AF). According to the results of bioassays, LPE has broad-spectrum activity against Gram-positive bacteria, Gram-negative bacteria and fungi. The mass spectrometry analysis of PLA revealed the existence of a novel compound that was named polycerradin. The purification of a novel antimicrobial peptide (AMP) from the AF was carried out by using chromatography. The compound was active against Gram-negative bacteria. Nterminal analysis determined the amino acid sequence, as well as MS / MS analysis confirmed the primary structure of this new compound. This research reports firstly the production of PAM PpRNCD that has an unusual amino acid in its constitution. It is an unprecedented fact considering the bacterial specie P. polymyxa. In terms of molecule size, PAM PpRNCD can be considered one of the smallest active natural peptide reported to date. It was also possible to isolate from FA the depsipeptides IL-F04a (m/z 883), LI-F04b (m/z 897), LI-F03a (m/z 947) and LI-F03b (m/z 961) previously described in the literature. The photoluminescence study of the LPE, TE, AF in both at room temperature (RT) and low temperature (T = 8K) was performed. In addition, this technique was applied to evaluate the action of the ELP on Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 29212, Shigella sonnei ATCC 1578 and Candida albicans ATCC 10231 in two different situations: (a) immediately after mixing LPE with the bacterial and fungus cell suspension, and (b) after thirty minutes. The photoluminescence emission was collected by a triple spectrometer (three diffraction gratings) T64000 model from Jobin Yvon, equipped with an optical microscope. For the detection of the radiation emitted by the sample we used a CCD camera (charge coupled device) cooled by liquid nitrogen. The slits of the spectrometer were adjusted to produce a spectral resolution of the order of 10-4 nm. The excitation source used was the line of 457 nm (violet) from an argon laser. The behaviors here observed indicate a strong potential for applications in biosensors as well as molecular markers. / Através do estudo de variáveis do cultivo pretendeu-se aumentar a produção de metabólitos antimicrobianos por Paenibacillus polymyxa RNC-D. Duas variáveis do processo - glicose e concentração de inóculo - foram avaliadas em diferentes níveis e seus efeitos na formação de biomassa, concentração inibitória mínima (CIM) contra Escherichia coli e redução na tensão superficial (RTS) foram estudados. Utilizando parâmetros não-otimizados as variáveis dependentes biomassa, CIM e RTS atingiram valores de 0,6 g/l, 1.000,0 μg/ml e 18,4 mN/m, respectivamente. As concentrações ótimas de glicose (16 g/l) e inóculo (5,0% v/v) foram definidas no sentido de maximizar a formação de biomassa e RTS do extrato, bem como diminuir o valor de CIM do extrato. Experimentalmente 2,05 g/l de biomassa; 31,2 μg/ml de CIM e 10,7 mN/m de RTS foram obtidos sob condições otimizadas. Foi constatado que altas concentrações do substrato glicose, embora refletissem em aumento de biomassa bacteriana, inibiram o metabolismo secundário microbiano, resultando em baixa produção de biomoléculas associada a altos valores de CIM. Através dos métodos de design fatorial experimental e superfície-resposta seguidos por otimização gráfica foi possível determinar a condição operacional ótima das concentrações iniciais de glicose e inóculo, as quais se demonstraram como variáveis de grande influência na produção de metabólitos antimicrobianos por P. polymyxa RNC-D. O extrato total (ET), proveniente do caldo de fermentação de P. polymyxa RNC-D, foi utilizado para pesquisa e isolamento de novos compostos antimicrobianos. Através de ensaios enzimáticos feitos com ET foi possível verificar a natureza lipopeptídica e peptídica dos compostos antimicrobianos. O ET foi submetido a um sistema de duas fases, separandose então em extrato lipopeptídico (ELP) e fração aquosa (FA). Resultados de bioensaios revelaram que o ELP apresenta amplo espectro de atividade contra bactérias Grampositivas, Gram-negativas e fungo. A análise por espectrometria de massas de ELP revelou a presença de um composto peptídico inédito o qual foi denominado polycerradin. A partir da fração aquosa (FA) foi possível a purificação de um novo peptídeo antimicrobiano (PAM) através de etapas cromatográficas. A bioatividade do composto foi avaliada e confirmada frente às bactérias Gram-negativas. A determinação da sequência de aminoácidos foi realizada por análise do N-terminal, e a confirmação da estrutura primária deste novo composto foi feita por MS/MS. O presente estudo relata pela primeira vez a produção do PAM PpRNCD que possui um aminoácido não usual em sua constituição, relato primeiramente aqui descrito considerando-se a espécie bacteriana P. polymyxa. Em termos de tamanho de molécula, pode-se considerar que o PAM PpRNCD é um dos menores peptídeos naturais ativos relatados até o momento. Utilizando-se a FA também foi possível o isolamento dos depsipeptídeos LI-F04a (m/z 883), LI-F04b (m/z 897), LI-F03a (m/z 947) e LI-F03b (m/z 961) previamente descritos na literatura. O estudo da fotoluminescência do ELP, do ET e da FA foi realizado tanto em temperatura ambiente (RT) quanto em baixa temperatura (T=8K). Também se estudou, através desta técnica, a ação do ELP sobre as bactérias Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 29212, Shigella sonnei ATCC 1578 e fungo Candida albicans ATCC 10231 em duas situações: (a) imediatamente após a mistura do ELP com a suspensão celular bacteriana, e (b) trinta minutos após a mistura. Detectou-se emissão fotoluminescente por ELP, ET e FA, e sinais de Raman a λ 699 nm (FA a baixa temperatura). Decorridos 30 min da mistura do ELP com as suspensões celulares microbianas houve alteração na emissão fotoluminescente, sendo que alguns sinais foram suprimidos (λ 470, 480 e 700 nm para S. sonnei, por exemplo). Isto evidencia a potencial aplicação destas frações (ELP, ET e FA) para a fabricação de sensores, detectores e marcadores moleculares.