Enterococcus faecalis 710C is a lactic acid bacterium that produces two bacterocins, ent7A and ent7B. Both ent7A and ent7B have strong activity against gram-positive food pathogens including Listeria spp., Clostridium spp., vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). Mass spectrometry analyses revealed that both ent7A (5201 Da) and ent7B (5207 Da) have formylated N-terminal methionine. The amino acid sequences, structural gene sequences of ent7 from nucleotide position 1-275 and immunity gene were determined. Circular dichroism data suggest that in aqueous solution ent7A and ent7B have 20 to 25% alpha-helical region. Addition of membrane-mimicking reagent (trifluoroethanol) did not significantly enhance the alpha-helical content in ent7A and ent7B. Chiral analysis by gas chromatography- mass spectrometry showed that the amino acid residues elucidated in ent7A and ent7B were all in L-configuration. / Food Science and Technology

Orientador: Eduardo Maffud Cillli / Banca: Ana Marisa Fusco Almeida / Banca: Vani Xavier de Oliveira Júnior / Resumo: Neste trabalho foi avaliado o efeito da dimerização e do comprimento/polaridade do espaçador utilizado na formação dos dímeros, na estrutura e atividade biológica do peptídeo antimicrobiano: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-Leu-CONH2. Nesse sentido, o monômero e três dímeros com diferentes espaçadores foram sintetizados pelo método de síntese de peptídeo em fase sólida (SPFS) utilizando uma combinação das químicas Fmoc e Boc. Análises por cromatografia de fase reversa e espectrometria de massas confirmaram o sucesso das sínteses e das purificações. Os ensaios de atividade antimicrobiana, em termos de CIM mostraram que a dimerização não aumentou a capacidade do peptídeo de inibir o crescimento de bactérias e fungos. No entanto, quando analisada a capacidade dos peptídeos de matar E. coli, um menor tempo para produzir o efeito inibitório foi observado para os dímeros. Adicionalmente, a atividade hemolítica dos peptídeos também foi avaliada, encontrando-se um aumento significativo, aproximadamente 40 vezes, para os dímeros em relação ao monômero. Com o objetivo de tentar explicar esses efeitos, o teste de proteção osmótica foi realizado. No entanto, o tamanho dos poros foi semelhante, o que não permite explicar as diferenças em termos desta variável. Conhecendo seus diâmetros, foi possível determinar que o poro é formado por 6 moléculas monoméricas ou 3 diméricas. Estudos de permeabilização mostraram que a porcentagem e a velocidade de liberação de carboxifluoresceína foram maiores para os dímeros quando comparados com o monômero, especialmente em vesículas contendo esfingomielina. Este ensaio também mostrou que a duplicação da concentração de monômeros não é suficiente para atingir a capacidade de permeabilização dos dímeros, confirmando que a proximidade das cadeias é um fator... (Resumo completo clicar acesso eletrônico abaixo) / Abstract: This work analyzed the effect of dimerization and the length/polarity of the spacer used in the formation of dimers, in the structure and biological activity of the antimicrobial peptide: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-Leu-CONH2. In this way, the monomer and three dimers (Lys-branched) with different spacer groups were synthesized by solid phase peptide synthesis methodology using a combination of Fmoc and Boc chemical approaches. Analysis by reverse phase chromatography and mass spectrometry confirmed the success of the synthesis and purifications. The antimicrobial activity assay, in terms of MICs showed that dimerization did not increase the ability of the peptides to inhibit the growth of bacteria and fungi. Nevertheless, when analyzing the peptides activity against E. coli in terms of kinetics, an increased velocity was observed for the dimers. The hemolytic activity of peptides was also evaluated, finding a very significant difference, approximately 40 times greater for dimers. Aiming to explain this difference, the osmotic protection test was performed, but the pore size was similar, which can not explain the differences in terms of this variable. Knowing the diameter of the pores, it was possible to determine that the pore is formed by six monomeric or three dimeric molecules. Additionally, permeabilization studies showed that the percentage and rate of carboxyfluorescein release were larger for the dimers compared with monomer, especially in vesicles containing sphingomyelin. This test also showed that the use of two times more monomer concentration's is not sufficient to reach dimers permeabilization capacity, confirming that the proximity of the chains is an important factor in the activity of this peptide. Analysis by circular dichroism revealed that peptides in aqueous solution are in random coil, whereas... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T19:49:48Z : No. of bitstreams: 1 nicolaslorenzon_e_me_araiq.pdf: 1141028 bytes, checksum: 6886093f73fd039fe1ed73b26a3d43dd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho foi avaliado o efeito da dimerização e do comprimento/polaridade do espaçador utilizado na formação dos dímeros, na estrutura e atividade biológica do peptídeo antimicrobiano: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-Leu-CONH2. Nesse sentido, o monômero e três dímeros com diferentes espaçadores foram sintetizados pelo método de síntese de peptídeo em fase sólida (SPFS) utilizando uma combinação das químicas Fmoc e Boc. Análises por cromatografia de fase reversa e espectrometria de massas confirmaram o sucesso das sínteses e das purificações. Os ensaios de atividade antimicrobiana, em termos de CIM mostraram que a dimerização não aumentou a capacidade do peptídeo de inibir o crescimento de bactérias e fungos. No entanto, quando analisada a capacidade dos peptídeos de matar E. coli, um menor tempo para produzir o efeito inibitório foi observado para os dímeros. Adicionalmente, a atividade hemolítica dos peptídeos também foi avaliada, encontrando-se um aumento significativo, aproximadamente 40 vezes, para os dímeros em relação ao monômero. Com o objetivo de tentar explicar esses efeitos, o teste de proteção osmótica foi realizado. No entanto, o tamanho dos poros foi semelhante, o que não permite explicar as diferenças em termos desta variável. Conhecendo seus diâmetros, foi possível determinar que o poro é formado por 6 moléculas monoméricas ou 3 diméricas. Estudos de permeabilização mostraram que a porcentagem e a velocidade de liberação de carboxifluoresceína foram maiores para os dímeros quando comparados com o monômero, especialmente em vesículas contendo esfingomielina. Este ensaio também mostrou que a duplicação da concentração de monômeros não é suficiente para atingir a capacidade de permeabilização dos dímeros, confirmando que a proximidade das cadeias é um fator... / This work analyzed the effect of dimerization and the length/polarity of the spacer used in the formation of dimers, in the structure and biological activity of the antimicrobial peptide: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-Leu-CONH2. In this way, the monomer and three dimers (Lys-branched) with different spacer groups were synthesized by solid phase peptide synthesis methodology using a combination of Fmoc and Boc chemical approaches. Analysis by reverse phase chromatography and mass spectrometry confirmed the success of the synthesis and purifications. The antimicrobial activity assay, in terms of MICs showed that dimerization did not increase the ability of the peptides to inhibit the growth of bacteria and fungi. Nevertheless, when analyzing the peptides activity against E. coli in terms of kinetics, an increased velocity was observed for the dimers. The hemolytic activity of peptides was also evaluated, finding a very significant difference, approximately 40 times greater for dimers. Aiming to explain this difference, the osmotic protection test was performed, but the pore size was similar, which can not explain the differences in terms of this variable. Knowing the diameter of the pores, it was possible to determine that the pore is formed by six monomeric or three dimeric molecules. Additionally, permeabilization studies showed that the percentage and rate of carboxyfluorescein release were larger for the dimers compared with monomer, especially in vesicles containing sphingomyelin. This test also showed that the use of two times more monomer concentration´s is not sufficient to reach dimers permeabilization capacity, confirming that the proximity of the chains is an important factor in the activity of this peptide. Analysis by circular dichroism revealed that peptides in aqueous solution are in random coil, whereas... (Complete abstract click electronic access below)

Resumo: A hepcidina é uma proteína que faz parte do sistema imune inato e desempenha um papel fundamental na regulação da homeostase do ferro. Este peptídeo foi previamente caracterizado em várias espécies, entretanto, até agora, não em ovinos. O objetivo deste estudo foi de determinar a sequência do RNAm codificante da hepcidina, bem como caracterizar e realizar a análise da expressão gênica deste peptídeo em diferentes tecidos de ovinos saudáveis. A região codificante da hepcidina consiste em 249 pares de base, as quais codificam um peptídeo contendo 82 aminoácidos. Este precursor, da forma bioativa da hepcidina, apresenta maior homologia com as sequências de Bos taurus e Bubalus bubalis. A hepcidina foi expressa predominantemente no fígado dos ovinos, contudo foram observados altos níveis de expressão no abomaso e baixos níveis nos outros tecidos. Estes resultados ampliam o conhecimento comparativo deste peptídeo, mostrando a relação da hepcidina ovina com a de outras espécies de mamíferos e será útil para estudos futuros sobre o metabolismo de ferro e do processo inflamatório nos ovinos. / Abstract: Hepcidin is part of the innate immune system, and plays a central role in the regulation of iron homeostasis. This peptide has been previously characterized in several species but not in sheep until now. The aim of this study was to sequence, characterize and perform hepcidin gene expression analysis in different tissues of healthy sheep. The resulting ORF consisted of 249 bp predicted to encode an 82 amino acids peptide. The deduced precursor was mostly homologous to Bos taurus and Bubalus bubalis. Hepcidin was predominantly expressed in liver, although high expression was present in abomasum and lower level expression occurred in other tissues. These findings extend our comparative knowledge of this peptide, showing the relationship between sheep hepcidin and other mammalian hepcidins and might be helpful for additional studies on iron metabolism and inflammatory process in sheep. / Orientador: Alexandre Secorun Borges / Coorientador: Paulo Eduardo Martins Ribolla / Banca: João Pessoa Araújo Júnior / Banca: Juliana Regina Peiró / Mestre

abstract: Random peptide microarrays are a powerful tool for both the treatment and diagnostics of infectious diseases. On the treatment side, selected random peptides on the microarray have either binding or lytic potency against certain pathogens cells, thus they can be synthesized into new antimicrobial agents, denoted as synbodies (synthetic antibodies). On the diagnostic side, serum containing specific infection-related antibodies create unique and distinct "pathogen-immunosignatures" on the random peptide microarray distinct from the healthy control serum, and this different mode of binding can be used as a more precise measurement than traditional ELISA tests. My thesis project is separated into these two parts: the first part falls into the treatment side and the second one focuses on the diagnostic side. My first chapter shows that a substitution amino acid peptide library helps to improve the activity of a recently reported synthetic antimicrobial peptide selected by the random peptide microarray. By substituting one or two amino acids of the original lead peptide, the new substitutes show changed hemolytic effects against mouse red blood cells and changed potency against two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. Two new substitutes are then combined together to form the synbody, which shows a significantly antimicrobial potency against Staphylococcus aureus (<0.5uM). In the second chapter, I explore the possibility of using the 10K Ver.2 random peptide microarray to monitor the humoral immune response of dengue. Over 2.5 billion people (40% of the world's population) live in dengue transmitting areas. However, currently there is no efficient dengue treatment or vaccine. Here, with limited dengue patient serum samples, we show that the immunosignature has the potential to not only distinguish the dengue infection from non-infected people, but also the primary dengue infection from the secondary dengue infections, dengue infection from West Nile Virus (WNV) infection, and even between different dengue serotypes. By further bioinformatic analysis, we demonstrate that the significant peptides selected to distinguish dengue infected and normal samples may indicate the epitopes responsible for the immune response. / Dissertation/Thesis / M.S. Biological Design 2013

Rapid development of antimicrobial resistance (AMR) among bacteria, combined with diminished new antibiotic discovery rates, is an increasing threat to human health. Bacterially derived antimicrobial peptides (AMP) hold excellent potential as potent novel therapeutics. This study embraces traditional natural AMP discovery methods and the newer in silico genome mining tool BAGEL 3 to facilitate identification of novel antimicrobial agents. The traditional screening efforts led to the discovery of two promising antimicrobial producer strains; Bacillus pumilus J1 producing two AMPs, peptides NI03 and NI04, and Klebsiella pneumoniae A7, which produced peptide NI05. In silico mining of the B. pumilus J1 and K. pneumoniae A7 genomes and those from under exploited anaerobic bacteria using BAGEL 3 yielded 18 putative bacteriocin structures that were associated with multiple known and relevant bacteriocin accessory genes and/or carried significant homologies to known bacteriocins. Peptide NI04 proved to be active against Gram positive species only, including meticillin resistant Staphylococcus aureus and vancomycin resistant enterococci and peptide NI03, in addition to these pathogens, showed activity against E. coli. Peptide NI05 was active against Gram-negative pathogens including extended spectrum β-lactamase producing E. coli. All isolated peptides were observed to be proteinaceous in nature and highly heat stable. Peptides were purified or partially purified using solid phase extraction followed by RP-HPLC. The mass of the peptides was determined using ESI or MALDI-TOF mass spectrometry. Tandem MS fragmentation of peptide NI04 generated several sequence tags. Draft genome sequences of the B. pumilus J1 and K. pneumoniae A7 producer strains were obtained using the Illumina MiSeq platform. This allowed identification of the genes encoding peptide NI04, which was confirmed to be novel and was named pumicin NI04. Further characterisation of pumicin NI04 demonstrated it was non-toxic to keratinocytes, Vero cells and non-haemolytic up to at least 18x the minimum inhibitory concentration. The discovery revealed that pumicin NI04 belongs to the WXG-100 peptide superfamily, having homology with the mycobacterial and staphylococcal virulence factor EsxA. This represents the first report of antimicrobial activity in a WXG-100 peptide and has intriguing evolutionary implications. Although it was not possible to fully characterise peptides NI03 and NI05, when BAGEL 3 was used to mine the B. pumilus J1 genome, a promising putative bacteriocin candidate was identified that was homologous to Enterocin AS-45, which also confers anti Gram-negative activity and may be related to the activity observed for NI03, however more evidence is required. Investigations of the K. pneumoniae A7 bacteriocin on the other hand helped establish that the K. pneumoniae microcin E492 pathway was present and highly conserved in strain A7, and is likely to be responsible for the activity observed indicating that NI05 was not a novel peptide.

Antimicrobial peptides (AMPs) have been identified as important therapeutic agents that can be developed as new multifunctional antibiotic compounds, which may address antibiotic resistance. AMPs have a wide range of bioactivities, including antimicrobial, antioxidant, anti-inflammatory and anticancer properties. Os and Os-C (a derivative of Os, lacking cysteine residues) are two synthetic AMPs derived from the tick defensin OsDef2 which have been shown to have antibacterial, antioxidant and anti-inflammatory activity. Differences in bacterial killing times between these peptides indicate differences in the modes of bacterial killing. For the further development of Os and Os-C for therapeutic application, the aim of this study was to establish the mode of bacterial killing, to determine if these peptides are cytotoxic to human erythrocytes and leukocytes. Lastly, to determine if these peptides have additional beneficial cellular effects such as antioxidant activity. Ultrastructural analysis with electron microscopy techniques revealed that both peptides adversely affected the membranes and intracellular structures of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria. Effects included membrane ruffling, cytoplasmic retraction, intracellular granulation and the formation of dense fibres. At the minimum bactericidal concentrations (MBCs) of 0.77 μM for Os and 1.74 μM for Os-C membrane permeabilisation measured with the SYTOX green assay was found not to be the principle mode of action. In stationary phase bacteria, fluorescent triple staining showed that both peptides caused permeabilisation. Studies using fluorescently labelled peptides revealed that the membrane penetrating activities of Os and Os-C were similar to buforin II, a cell-penetrating peptide. Os was able to enter stationary phase E. coli and B. subtilis while Os-C was unable to enter E. coli cells and accumulated on B. subtilis septa. Using plasmid binding and fluorescence displacement assays both peptides could bind DNA, while a dosage effect was only observed for Os. Evaluation of cytotoxicity revealed that Os and Os-C caused no erythrocyte haemolysis or changes to erythrocyte morphology. Only the highest concentration of Os (100 μM), which is 130 fold greater than the MBC for E. coli and B. subtilis, caused cellular damage to peripheral mononuclear (MN) and polymorphonuclear (PMN) cells. In contrast, Os-C caused leukocyte activation identified by associated morphological features and reactive oxygen species (ROS) formation. Chemical and erythrocyte antioxidant assays indicated that both Os and Os-C had antioxidant activity. Both peptides provided extracellular protection of erythrocytes against 2,2'-azobis(2-amidinopropane) dihydrochloride induced oxidative damage. In MN and PMN cells Os showed low levels of antioxidant activity while Os-C had minimal activity. In conclusion, both peptides showed a dual mechanism of bacterial killing, targeting both the membrane and intracellular elements. Os had a predominant membrane effect while Os-C targeted the septa of B. subtilis and had a higher affinity for DNA. Cytotoxicity in erythrocytes and leukocytes was minimal. In addition, Os exhibited antioxidant properties while Os-C caused leukocyte activation. Both peptides have been identified as promising therapeutic agents although activity in plasma and the effect on coagulation must still be determined. / Thesis (PhD)--University of Pretoria, 2017. / Anatomy / PhD / Unrestricted

The isolation, purification, and clinical deployment of antibiotics is one of the major drivers of decrease in morbidity and mortality from infectious bacteria in the 20th century. The rapid, ubiquitous deployment of antibiotics encouraged swift development and distribution of antibiotic resistance. New, novel techniques, technologies, and ultimately therapeutic antimicrobial compounds will be required to counter the rise of antibiotic resistant microbes. Historically, mimicking naturally occurring compounds has been the most fruitful method for discovering new antibiotics; unsurprisingly, many recent efforts have focused on expanding the cultivation and detection of previously unknown microbes and compounds, respectively. Other techniques explore developing compounds de novo, reverse-engineering potential therapies from a detailed understanding of the biochemistry of pathogens. We describe a novel peptide screening tool in E. coli designed to be used for such an application. This platform, termed PepSeq, is capable of screening millions of peptides simultaneously by using Illumina sequencing technology. Additionally, we have explored several peptide scaffolds that have a conserved secondary structure with a large randomizable domain of several amino acids, which allows the screening for new and novel biochemical interactions with more stable structure than a simple linear peptide. Finally, we have developed a bioinformatics workflow that complements PepSeq that allows analysis of PepSeq data for peptide motifs of interest, vastly streamlining motif identification and verification.

The isolation, purification, and clinical deployment of antibiotics is one of the major drivers of decrease in morbidity and mortality from infectious bacteria in the 20th century. The rapid, ubiquitous deployment of antibiotics encouraged swift development and distribution of antibiotic resistance. New, novel techniques, technologies, and ultimately therapeutic antimicrobial compounds will be required to counter the rise of antibiotic resistant microbes. Historically, mimicking naturally occurring compounds has been the most fruitful method for discovering new antibiotics; unsurprisingly, many recent efforts have focused on expanding the cultivation and detection of previously unknown microbes and compounds, respectively. Other techniques explore developing compounds de novo, reverse-engineering potential therapies from a detailed understanding of the biochemistry of pathogens. We describe a novel peptide screening tool in E. coli designed to be used for such an application. This platform, termed PepSeq, is capable of screening millions of peptides simultaneously by using Illumina sequencing technology. Additionally, we have explored several peptide scaffolds that have a conserved secondary structure with a large randomizable domain of several amino acids, which allows the screening for new and novel biochemical interactions with more stable structure than a simple linear peptide. Finally, we have developed a bioinformatics workflow that complements PepSeq that allows analysis of PepSeq data for peptide motifs of interest, vastly streamlining motif identification and verification.

This thesis presents applications of high resolution secondary ion mass spectrometry (NanoSIMS) analysis for stable isotope imaging in biological samples. These projects were designed to explore the potential applications of NanoSIMS analysis, and to develop protocols and novel methodologies to visualize and quantify biological processes. Working with collaborators in the UK and USA, I have applied NanoSIMS analysis to study 3 research areas, including molecule interactions, single cell metabolisms and lipid imaging in tissues. Antimicrobial peptides (AMPs) play important role in the immune system, and understanding how AMPs interact with cell membranes can provide useful information to design new therapies to control infection. The pore structures and dynamics of the interaction of AMPs with membranes has been visualized for the first time and confirmed with combined AFM and NanoSIMS analysis. A correlative backscattered electron (BSE) imaging and NanoSIMS analysis methodology has been developed to study glutamine metabolism in single cancer cells. This method enables us to measure the chemical information in specific organelles in these cells and can be widely applied to study metabolisms and to trace the uptake of labelled molecules in biological matrices. Quantitative analysis on the effects of hypoxic conditions and the PYGL gene were studied. Applying correlative BSE and NanoSIMS analysis, I also studied lipid uptake mechanisms in various mouse tissues, including brown adipose tissue, heart, intestines, liver and skeletal muscle, mainly focused on a recently discovered protein, GPIHBP1, and its function in the lipid uptake process. TRL margination was proved to depend on the GPIBP1-LPL complex, and 3 stages of lipid transport from capillary lumen to lipid droplets was also visualized by combined BSE and NanoSIMS analysis.