Antimicrobial peptides (AMPs) are found throughout the animal kingdom and act as a natural defence against a broad spectrum of pathogens. These peptides are toxic to invading organisms without acting on host cells, so are of interest for their potential to act as potent new drugs against pathogenic organisms. AMPs traverse the cell wall and predominantly target the plasma membrane, resulting in destabilisation, leakage of intracellular components and cell death. In this thesis the mode of action of several AMPs was investigated. The role of the cell wall was studied and found to mediate peptide binding, the inhibition of certain cell wall components also increased peptide action, subsequent internalisation events were observed with varying localisation patterns and the effect of several genes that alter cell susceptibility to AMP were examined. Several Candida albicans mutants, each deficient in cell wall protein mannosylation, were tested in relation to their susceptibility to AMPs. It was discovered that cells lacking or deficient in the phosphomannan fraction, with a concomitant reduction in surface negative charge, correlated with reduced susceptibility to AMP action. To ascertain whether peptide binds to negatively charged phosphate, the effect of exogenous glucosamine 6-phosphate (but not glucosamine hydrochloride) was studied demonstrating that peptide efficacy was reduced due to the presence of exogenous phosphate. More specifically, sequestration of the truncated cationic AMP dermaseptin S3 (DsS3(1-16)) was reduced in these phosphomannan deficient mutants. Microscopy analysis of fluorescein tagged DsS3(1-16) also revealed the differential localisation patterns of this AMP: transiently binding to the plasma membrane, localisation to the vacuole or diffuse distribution throughout the cytoplasm. It is proposed that for these cationic AMPs to exert their full antifungal action they must first bind to the negatively charged phosphate. The echinocandins are a relatively new class of antifungal that function by inhibiting 1,3-β glucan synthase resulting in reduced 1,3-β glucan in the cell wall. As AMPs have to traverse the cell wall it was postulated that cells lacking this fraction would display increased AMP binding to the membrane. Clinical isolate strains of Candida and Cryptococcus spp. were acquired to test their susceptibility to AMP and echinocandin combinations. Comparing the fractional inhibitory concentration index (FICI) (supported by viable cell counts and on a solid surface using disc diffusion assays) synergy was observed between caspofungin, anidulafungin and several AMPs in vitro. In vitro toxicity assays revealed no increase in haemolytic or cytotoxic action on combination. These synergistic combinations could provide a novel treatment against fungal pathogens. The final area of study was based upon work that identified genes whose expression altered cell susceptibility to AMPs. Three genes were selected for investigation that upon deletion increased the action of DsS3(1-16) or magainin 2 on S. cerevisiae. Results from growth analysis, peptide sequestration and cell viability counts confirmed that deletion of HAL5, LDB7 or IMP2’ did increase susceptibility. Additionally, deletion of HAL5 increased the probability of cell depolarisation upon peptide exposure. Expression of GFP-tagged Imp2’ also increased when cells were exposed to DsS3(1-16). It was concluded that deletion of HAL5 increases depolarisation due to insufficient potassium efflux, leading to ion leakage and cell death facilitated by AMP action. Double strand break repair and DNA protection are probably compromised upon deletion of LDB7 and IMP2’, increasing the inhibitory action of DsS3(1-16) that has previously been shown to bind to DNA.

Les bactériocines sont des peptides antimicrobiens produits par des bactéries naturellement immunisées contre leurs propres bactériocines. Ce travail a permis l’identification de nombreuses souches productrices de peptides antimicrobiens. Ces derniers ont été extraits et purifiés par différentes techniques chromatographiques, puis identifiés et caractérisés par la mesure de leurs masses et par l’analyse de leurs structures (approche protéomique et séquençage d’Edman). Parmi les résultats obtenus, il y a :- la mise en évidence d’une nouvelle bactériocine (maltaricin CPN), appartenant à la classe IIa, isolée et identifiée chez Carnobacterium maltaromaticum ;- l’identification de trois nouvelles espèces productrices de pédiocine PA-1 ;- l’isolement de souches productrices de bactériocines multiples (appartenant à différentes classes) ;- la mise en évidence pour certaines bactériocines d’une forte activité antimicrobienne in vitro (spectres d’activité incluant des pathogènes).Les travaux se poursuivent avec l’application de deux souches productrices de bactériocines dans du lait fermenté (Lben) en vue de lutter contre L. monocytogenes.Les bactériocines sont des peptides antimicrobiens produits par des bactéries naturellement immunisées contre leurs propres bactériocines. Ce travail a permis l’identification de nombreuses souches productrices de peptides antimicrobiens. Ces derniers ont été extraits et purifiés par différentes techniques chromatographiques, puis identifiés et caractérisés par la mesure de leurs masses et par l’analyse de leurs structures (approche protéomique et séquençage d’Edman). Parmi les résultats obtenus, il y a :- la mise en évidence d’une nouvelle bactériocine (maltaricin CPN), appartenant à la classe IIa, isolée et identifiée chez Carnobacterium maltaromaticum ;- l’identification de trois nouvelles espèces productrices de pédiocine PA-1 ;- l’isolement de souches productrices de bactériocines multiples (appartenant à différentes classes) ;- la mise en évidence pour certaines bactériocines d’une forte activité antimicrobienne in vitro (spectres d’activité incluant des pathogènes).Les travaux se poursuivent avec l’application de deux souches productrices de bactériocines dans du lait fermenté (Lben) en vue de lutter contre L. monocytogenes. / Bacteriocins are antimicrobial peptides produced by bacteria naturally immunized against their own bacteriocins. This work has allowed the identification of several strains which produce antimicrobial peptides. These have been purified using different chromatographic techniques. Then, they have been identified and characterized by the measurement of their mass and by the analysis of their structure (proteomic approach/ Edman sequencing). Among the obtained results, there was:- the discovery of a new bacteriocin (maltaricin CPN) produced by C. maltaromaticum and belonging to the class IIa ;- the identification of three new pediocin PA-1 producing species;- the isolation of bacterial strains wich produce multiple bacteriocins belonging to several classes ;- the in vitro determination of a strong antimicrobial activity(affecting pathogens) with some bacteriocins.This work is still underway with the application of two bacteriocin producing strains in fermented milk (Lben) in order to tackle L. monocytogenes.

We identified a synthetic hexapeptide that blocks Magnaporthe grisea appressorium formation, in artificial hydrophobic surface. The results suggest that peptides interfere with surface recognition. M. grisea non pathogenic pth1 mutants were complemented by N. crassa orthologous gene suggesting that the biochemical function of pth1 has not evolved specifically to play a role in appressorium development.

Most gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.

To explore the use of defensins for transgenic plant disease resistance, expression by agroinfiltration of plants, stable transformation of plants and stable transformation of yeast were tested for porcine β-defensin 1 (pbd-1) and Nicotiana tabacum defensin 1 (Ntdef1). Attempts to screen constructs by agroinfiltration of Nicotiana benthamiana leaves revealed that agroinfiltration alone induced localized resistance against Colletotrichum destructivum. A comparison of Agrobacterium tumefaciens strains showed that the induced resistance required the transfer of type IV effectors into plant cells and was independent of salicylic acid or ethylene signaling. Stable expression of pbd-1 in N. tabacum and Pichia pastoris showed that PBD-1 purified from P. pastoris had varying degrees of antimicrobial activity against a broad range of microbes, including P. syringae pv. tabaci, C. destructivum and C. orbiculare, but in transgenic N. tabacum, the protein could not be detected and resistance increased only slightly to P. syringae pv. tabaci but not to C. destructivum or C. orbiculare. Stable expression of Ntdef1 in P. pastoris yielded a protein with no or little antimicrobial activity, and stable expression in N. tabacum did not result in detectable Ntdef1 or increased resistance to those pathogens. Although PBD-1 had strong antimicrobial activity against plant pathogens, plant disease resistance likely did not increase because of the low level of the protein in the plants, whereas resistance did not increase with Ntdef1 likely because of low antimicrobial activity and low levels of the protein in the plant. This research demonstrates that agroinfiltration is not appropriate for testing genes for antimicrobial activity in planta, while the P. pastoris expression system is useful for producing protein for in vitro tests of a gene prior to its transfer to plants.

Orientador: Samira Esteves Afonso Camargo / Banca: Luana Marotta Reis de Vasconcellos / Banca: Mônica Ghislaine Oliveira Alves / Banca: Cristina Pacheco Soares / Banca: Cacio de Moura Netto / Resumo: O peptídeoLL-37 (catelicidina derivada de humano), é liberado por algumas células humanas e capaz de neutralizar os tecidos com lipopolissacarídeo (LPS), além de atrair células da polpa, e induzir a angiogênese, características que o tornam um possível adjunto para a regeneração do complexo dentino-pulpar. O objetivo desse trabalho foi avaliar in vitro a biocompatibilidade do peptídeo LL-37 nas concentrações de 5 e 10 µg/mL, e sua possível atuação na diferenciação de células-tronco da polpa dentária (DPSC) para odontoblastoslike. Com esse propósito, foram avaliados: (a) a citotoxicidade, pelo teste MTT; (b) a genotoxicidade, através do ensaio do micronúcleo; (c) a produção e quantificação de óxido nítrico; (d) as fases do ciclo celular, por citometria; (e) a expressão de alguns genes associados à formação de tecido mineralizado, através do teste qRT-PCR; (f) o conteúdo de proteína total; (g) a atividade de fosfatase alcalina (ALP); e (h) a produção de sialofosfoproteína dentinária (DSPP), pelo ensaio imunoenzimático ELISA. Foi observado que as concentrações de 5 e 10 µg/mL de LL-37 não foram citotóxicas e ainda aumentaram, em geral, a viabilidade celular (p<0,05), sendo que os maiores valores de absorbância foram observados no 3° dia de contato. As concentrações testadas também não induziram genotoxicidade, após 7 dias de contato, tendo sido genotóxico apenas o grupo controle positivo (EMS) (p<0,05). Ainda, não foi observado diferença estatisticamente significativa na produção de nitrito, pelas células expostas ao LL-37 após 7 dias, em ambas as concentrações. A análise do ciclo celular, evidenciou maior porcentual de células na fase G0/G1, em todos os grupos (p<0,05). Quando estes foram comparados, foi observado maior quantidade de células na fase G0/G1 na concentração de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract : The LL 37 peptide (human derived cathelicidin) is released by some human cells and able of neutralizing the tissues that present lipopolysaccharide (LPS), as well as, attracts pulp cells and induces angiogenesis; characteristics that makes it a possible adjunct for regeneration of the dentin-pulp complex. The aim of this study was evaluate in vitro the biocompatibility of LL-37 in the concentrations of 5 and 10 µg/mL, and its possible performance in the differentiation of dental pulp stem cells (DPSC) into odontoblasts-like cells. For this purpose, it was evaluated: (a) the cytotoxicity by MTT assay; (b) the genotoxicity by the micronucleus test; (c) the production and quantification of nitric oxide; (d) the cell cycle, by flow cytometry; (e) the expression of genes associated with the mineralization by qRT-PCR; (f) the total protein content; (g) the alkaline phosphatase activity (ALP); and (h) the production of dentine sialofosfoprotein (DSPP) by indirect enzyme-linked immunosorbent assay (ELISA). It was observed that the concentrations of 5 and 10 µg/ml of LL-37 were not cytotoxic, in addition to they increased, in general, the cell viability (p<0,05). Moreover, higher absorbance values were observed on 3rd day of contact. After 7 days, the tested concentrations also did not induce genotoxicity, (p<0,05); only the positive control group (EMS) was genotoxic (p<0.05). Furthermore, there was not statistical significance in the nitrite production by the cells exposed to LL-37 for 7 days, in both concentrations. The cell cycle test showed higher percentage of cells in the phase G0/G1 in all groups (p<0.05). When they were compared, it was noticied that concentration of 10 ug/ml of LL-37 arrested the cells in G0/G1 compared to the control group (p<0.05). On the other hand, the control group, exhibited higher amount of cells in G2 and mitosis...(Resumo completo, clicar acesso eletrônico abaixo) / Doutor

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A cÃrie dental à conceituada como uma doenÃa infectocontagiosa, crÃnica e multifatorial na qual ocorre uma desmineralizaÃÃo progressiva das estruturas dentais com consequente dor e perda do elemento dental. à considerada um problema de saÃde pÃblica em todo mundo devido sua incidÃncia e consequÃncias orais e sistÃmicas. Dessa forma, novos mÃtodos de controle microbiano tÃm sido pesquisados com vista à reduÃÃo do nÃmero de casos. Os peptÃdeos antimicrobianos sÃo molÃculas presentes em diversos seres vivos e possuem uma alta atividade biocida frente a diversos microrganismos patogÃnicos. O peptÃdeo Lys-[Trp6]-Hy-A1 (Lys-a1), derivado sintÃtico do peptÃdeo Hy-A1, isolado inicialmente da espÃcie Hypsiboas albopunctatus, à uma molÃcula com atividade antimicrobiana descrita na literatura. Dessa forma, o objetivo deste trabalho foi avaliar o potencial antibacteriano do peptÃdeo sintÃtico Lys-a1 sobre crescimento planctÃnico e em biofilme de bactÃrias orais. As metodologias utilizadas para avaliaÃÃo do potencial antimicrobiano foram: determinaÃÃo da concentraÃÃo inibitÃria mÃnima (CIM) e concentraÃÃo bactericida mÃnima (CBM) em placas de poliestireno para o crescimento em suspensÃo; e quantificaÃÃo de biomassa por cristal violeta e contagem de unidades formadoras para crescimento em biofilme. Os micro-organismos, S. oralis, S. sanguinis, S. parasanguinis, S. salivarius, S. mutans e S. sobrinus, foram cultivados em Brain Heart Inffusion caldo suplementado com 1% de sacarose (BHIs) a 37 ÂC sob atmosfera com 10% de CO2. O peptÃdeo foi solubilizado em Ãcido acÃtico 0,1% (v/v) em diferentes concentraÃÃes (500 a 1,9 Âg.mL-1). Os grupos controle dos ensaios foram meio de cultura BHIs (controle negativo) e Gluconato de Clorexidina 0,12% (controle positivo). O peptÃdeo testado apresentou um destacado efeito antimicrobiano, sendo capaz de inibir o crescimento planctÃnico e em biofilme de todas as cepas testadas mesmo em baixas concentraÃÃes. Assim, o peptÃdeo Lys-a1 à uma importante fonte para possÃveis agentes antimicrobianos, com Ãnfase no controle e prevenÃÃo de biofilmes microbianos, um dos fatores mais importantes para o desenvolvimento do processo cariogÃnico. / Dental caries is defined as an infectious, chronic and multifactorial disease, in which there is a progressive demineralization of tooth structure with consequent pain and dental loss. It is considered a major public health problem worldwide because its high incidence, besides its oral and systemic consequences. Thus, new methods of microbial control have been investigated to reduce the number of cases. Antimicrobial peptides are molecules present in many living beings and have a high biocidal activity against various pathogenic microrganisms. The peptide Lys-[Trp6]-Hy-A1 (Lys-a1) is a synthetic derivative of the peptide Hy-A1, initially isolated from the species Hypsiboas albopunctatus. According to previous research, it is a molecule with broad antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of the synthetic peptide Lys-a1 on the planktonic and biofilm growth of oral bacteria. The methods used to evaluate antimicrobial activity include the following: determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in microtiter plates for growth in suspension and quantification of biomass by crystal violet staining and counting of colony forming units for biofilm growth. The microorganisms S. oralis, S. sanguinis, S. parasanguinis, S. salivarius, S. mutans and S. sobrinus were grown in Brain Heart Infusion broth supplemented with 1% sucrose (BHIs) at 37 ÂC under atmospheric pressure with 10% CO2. The peptide was solubilized in 0.1% acetic acid (v/v) at various concentrations (500 to 1.9 Âg.mL-1). Chlorhexidine gluconate 0.12% was used as the positive control, and BHIs culture medium was used as the negative control. The tested peptide demonstrated a remarkable antimicrobial effect, inhibiting the planktonic and biofilm growth of all strains tested, even at low concentrations. Thus, the peptide Lys-a1 is an important source for potential antimicrobial agents, especially for the control and prevention of microbial biofilms, which is one of the most important factors in cariogenic processes.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / A maioria dos micro-organismos encontra-se integrados em comunidades denominadas âbiofilmeâ. Dentre estes se destaca a Pseudomonas aeruginosa, um patÃgeno oportunista de vegetais, animais e humanos, frequentemente associado à infecÃÃes nosocomiais difÃceis de serem erradicadas devido à presenÃa de biofilme. Os antibiÃticos fluoroquinolonas, tais como a ciprofloxacina (CIP), sÃo utilizados contra esta bactÃria, entretanto, mecanismos de resistÃncia vem sendo desenvolvidos por este micro-organismo. Assim, novas formas de controle microbiano tÃm sido alvo de inÃmeras pesquisas a fim de substituir ou potencializar os mÃtodos convencionais. Para esse fim, peptÃdeos antimicrobianos (AMPs) obtidos de diversos seres vivos representam uma alternativa para o desenvolvimento de novas drogas. Formas sintÃticas anÃlogas ao AMP Hilina a1 (Hy-a1), isolado da espÃcie Hypsiboas albopunctatus, tem demonstrado excelente eficÃcia antimicrobiana, dentre os quais se destacam o peptÃdeo Lys-[Trp6]hy-a1 (Lys-a1). Dessa forma, o objetivo deste trabalho foi verificar a influÃncia do peptÃdeo Lys-a1 na atividade antimicrobiana de CIP contra o micro-organismo patogÃnico Pseudomonas aeruginosa ATCC 9027. A concentraÃÃo inibitÃria mÃnima (CIM), a concentraÃÃo bactericida mÃnima (CBM) e a curva de morte foram determinadas para a CIP e a Lys-a1, separadamente. A influÃncia da Lys-a1 na atividade antimicrobiana da CIP foi verificada pela metodologia checkerboard com quantificaÃÃo da biomassa e do nÃmero de cÃlulas viÃveis do biofilme atravÃs da coloraÃÃo pelo cristal violeta e contagem de unidades formadoras de colÃnia (UFC), respectivamente. Os valores de CIM e CBM da Lys-a1 foi de 125 &#956;g.mL-1, e para a CIP foi de 0,24 e 0,48 &#956;g.mL-1, respectivamente. Os dados mostraram que tanto a CIP quanto a Lys-a1 sÃo potentes antimicrobianos contra P. aeruginosa, sendo capazes de inibir tanto o crescimento planctÃnico como o desenvolvimento do biofilme. A interaÃÃo das substÃncias foi interpretada pelo Ãndice de concentraÃÃo inibitÃria fracional (ICIF) e mostrou um efeito aditivo, estabelecendo uma diminuiÃÃo de 16 vezes da CIM e da CBM da CIP, quando combinada com a Lys-a1. Esta influÃncia foi observada tambÃm na cinÃtica de morte e na atividade antibiofilme. Em conclusÃo, o efeito aditivo entre a Lys-a1 e a CIP na atividade antimicrobiana sugere que o peptÃdeo tem potencial como um agente terapÃutico e adjuvante para o tratamento de doenÃas infecciosas causadas por P. aeruginosa, podendo habilitar o uso de concentraÃÃes menores do antibiÃtico. / Most microorganism lies embedded in communities called "biofilms". Among these stand out Pseudomonas aeruginosa, an opportunistic pathogen of plants, animals and humans, frequently associated with nosocomial infections difficult to be eradicated due to the presence of biofilm. The fluoroquinolone antibiotics such as ciprofloxacin (CIP) against this bacterium are used, however, the mechanism of resistance has been developed for this microorganism. Thus, new forms of microbial control have been the subject of numerous studies in order to replace or enhance conventional methods. For this purpose, antimicrobial peptides (AMPs) obtained from various living beings represent an alternative to the development of new drugs. Synthetic forms analogous to Hylin a1 AMP (Hy-a1), isolated from species Hypsiboas albopunctatus, has shown excellent antimicrobial efficacy, among which stand out the peptide Lys-[Trp6]hy-a1 (Lys-a1). Thus, the aim of this work was to verify the influence of the peptide Lys-a1 in antimicrobial activity of CIP against pathogenic microorganism Pseudomonas aeruginosa ATCC 9027. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the curve death were determined to CIP and Lys-a1 separately. The influence of Lys-a1 in the antimicrobial activity of the CIP has been verified by the checkerboard method to quantify the biomass and the number of viable cells in biofilms by crystal violet staining and counting of colony forming units (CFU), respectively. The MIC and MBC values of Lys-a1 was 125 &#956;g.mL-1 and the CIP was 0.24 and 0.48 &#956;g.mL-1, respectively. The data showed that both the CIP as Lys-a1 are potent antibiotics against P. aeruginosa is able to inhibit both the planktonic growth and biofilm development. The interaction of substances interpreted by the fractional inhibitory concentration index (FICI) and showed an additive effect, establishing a decrease of 16 times the MIC and MBC of the CIP, when combined with the Lys-a1. This effect was also observed in the kinetics of death and antibiofilm activity. In conclusion, the additive effect of the Lys-a1 and CIP in antimicrobial activity suggests that the peptide has potential as a therapeutic agent and an adjuvant for the treatment of infectious diseases caused by P. aeruginosa can enable the use of lower concentrations of the antibiotic.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-02-16T14:50:28Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Thesis_Gabriel_digital.final.pdf: 10412972 bytes, checksum: ec85b3671bb5bbcd735ea9e83436b08f (MD5) / Made available in DSpace on 2017-02-16T14:50:28Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Thesis_Gabriel_digital.final.pdf: 10412972 bytes, checksum: ec85b3671bb5bbcd735ea9e83436b08f (MD5) Previous issue date: 2016-04-01 / CNPq / Peptídeos antimicrobianos são componentes importantes do sistema de defesa de diversos organismos contra possíveis invasores. Em geral, são pequenos (até 100 aminoácidos), catiônicos e anfipáticos. Eles têm despertado o interesse da comunidade científica por sua capacidade de atuação contra micróbios, que não conseguem desenvolver resistência a esses peptídeos. Ou seja, eles emergem como complemento e/ou alternativa ao uso dos antibióticos convencionais. Este trabalho desenvolveu um modelo computacional de um peptídeo híbrido de pediocina A (N-terminal) e plantaricina 149a (C-terminal), dois peptídeos bactericidas. Dados experimentais obtidos pelo grupo da prof. Dra. Rosângela Itri do IFUSP foram utilizados para modelagem e comparação dos resultados. Foram feitas simulações de MD do peptídeo interagindo com membranas puras e mistas de POPC e POPG utilizando os parâmetros do campo de força GROMOS 53A6 e 54A7. As simulações com uma unidade do peptídeo revelaram a atualização 54A7 era a mais adequado para modelagem desses sistemas. Os mapas de estrutura secundária mostraram que o peptídeo adquire configuração mais ordenada quando interage com membranas com maior quantidade de POPG em sua composição. As simulações com duas unidades do peptídeo sugeririam que o peptídeo interage e penetra na camada de POPG através da região Cterminal. Na simulação com membrana de POPC, nenhuma das porções terminais ficou estável no interior da membrana. O efeito do aumento da concentração de peptídeos foi examinado colocando cinco e dez unidades do peptídeo para interagir com as membranas. Na membrana de POPC, os peptídeos não formam um único aglomerado e causam pouca perturbação na bicamada. Já na membrana de POPG, o efeito da interação do aglomerado de peptídeos é acentuado, provocando grandes deformações na bicamada lipídica, praticamente a destruindo. Esse fenômeno sugere um possível mecanismo carpete para ação do peptídeo sobre a membrana fosfolipídica de bactérias. / Antimicrobial peptides are important components of defense system in various organizations against possible invaders. They are generally small (100 aminoacids), cationic and amphipathic. They have stimulated the interest of the scientific community for its ability to act against microbes that cannot develop resistance to these peptides. That is, they emerge as complement and/or alternative to the use of conventional antibiotics. This study developed a computational model of a hybrid peptide pediocin A (Nterminal) and plantaricin 149a (C-terminal), two bactericidal peptides. Experimental data obtained by the group of prof. Dr. Rosângela Itri (IFUSP) were used for modeling and compare the results. MD simulations were made of the peptide interacting with pure and mixed POPC and POPG membranes. These simulations were performed using the parameters of the force field GROMOS 53A6 and 54A7. Simulations with a single copy of the peptide revealed that the force field 54A7 was the most appropriate for modeling these systems. The secondary structure maps showed that the peptide acquires a more ordered configuration when interacting with membranes with higher amounts of POPG in its composition. The simulations with two copies of the peptide suggest that the peptide interacts and penetrates the POPG layer via the C-terminal part. In the simulation with POPC membrane, none of the end portions remained stable within the membrane. The effect of increasing the peptide concentration of was examined by placing five and ten copies of the peptide to interact with the membranes. In the POPC membrane, the peptides do not form a single cluster and they cause little disturbance in the bilayer. In the POPG membrane, the interaction of peptides cluster is enhanced, causing large deformation and practically destroying the lipid bilayer. This phenomenon suggests a possible carpet mechanism of action of the peptide on the phospholipid membrane of bacteria.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-04T11:57:30Z No. of bitstreams: 1 leticiastephantavares.pdf: 1145142 bytes, checksum: 8dd69d31541451cdce69de313d2a2aa6 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-04T15:35:32Z (GMT) No. of bitstreams: 1 leticiastephantavares.pdf: 1145142 bytes, checksum: 8dd69d31541451cdce69de313d2a2aa6 (MD5) / Made available in DSpace on 2017-04-04T15:35:32Z (GMT). No. of bitstreams: 1 leticiastephantavares.pdf: 1145142 bytes, checksum: 8dd69d31541451cdce69de313d2a2aa6 (MD5) Previous issue date: 2009-03-12 / A busca por novos antibióticos de amplo espectro de ação tem aumentado nas últimas décadas devido ao número crescente de bactérias resistentes aos antibióticos convencionais. O peptídeo recombinante Pg-AMP1, expresso em um sistema heterólogo em Escherichia coli, apresentou atividade antibacteriana contra linhagens Gram-positivas e Gram-negativas. A partir da seqüência de aminoácidos do peptídeo isolado de sementes de goiaba (Psidium guajava L.) o gene correspondente ao peptídeo foi modificado utilizando-se o códon preferencial para expressão em E. coli e construído um vetor de expressão. Uma região codificadora para cauda de histidina foi fusionada ao gene pg-amp1 permitindo a purificação por cromatografia de afinidade com íons de níquel imobilizados em coluna de sefarose. Utilizando SDS-PAGE e análise in sílico identificamos a massa molecular do PgAMP1 recombinante de 7,368 kDa e pI 8.93. O peptídeo recombinante foi expresso principalmente na forma insolúvel, agregando-se em corpos de inclusão que foram tratados com agentes desnaturantes obtendo o peptídeo solúvel. Após a purificação pela coluna de níquel obtivemos um rendimento de 13 mg por litro de meio de cultura. O peptídeo Pg-AMP1 recombinante apresentou atividade contra as bactérias Gram-negativas Escherichia coli e Pseudomonas aeruginosa e contra as Grampositivas Staphylococcus aureus e Staphylococcus epidermides. O peptídeo recombinante Pg-AMP1 não mostrou atividade contra os fungos fitopatogênicos testados. Devido a sua ação contra estas cepas de bactérias patogênicas humanas, o Pg-AMP1 recombinante torna-se um promissor antimicrobiano a ser utilizado no desenvolvimento de novos antibióticos contra cepas resistentes aos fármacos comumente usados. / The search for new antibiotics of broad spectrum of activity has increased in recent decades due to the increasing number of bacteria resistant to conventional antibiotics. The Pg-AMP1 recombinant peptide expressed in a heterologous system in Escherichia coli, strains showed antibacterial activity against Gram-positive and Gram-negative. From the sequence of amino acid peptide isolated from the seeds of guava (Psidium guajava L.) peptide corresponding to the gene was modified using the preferred codon for expression in E. coli and constructed a vector for expression. A region coding for the histidine tail was merged the gene-pg amp1 allowing purification by affinity chromatography with nickel ions immobilized on the column sepharose. Using SDS-PAGE and in silico analysis identified the molecular weight of Pg-AMP1 recombinant with 7.368 kDa and pI 8.93. The recombinant peptide was expressed mostly as insoluble form, adding up in inclusion bodies, which were treated with denaturants agents to solubilize the peptide. The purification of peptide by nickel sepharose column yielded 13 mg per liter of culture medium. The Pg-AMP1 recombinant peptide showed activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and against gram-positive Staphylococcus aureus and Staphylococcus epidermidis. The recombinant peptide Pg-AMP1 showed no activity against the phytopathogenic fungi tested. Due to its action against these strains of human pathogenic bacteria, the recombinant Pg-AMP1 is a promising antimicrobial for use in developing new antibiotics against strains resistant to commonly used drugs.