Since ticks are classified as hematophagous ectoparasites, the primary feeding event involves a bloodmeal on a vertebrate host. Such activities facilitate the ingestion of microorganisms which may be detrimental to the survival of a tick. It is observed, however, that ticks are able to survive such invasion by microorganisms and in several cases, facilitate the transmission of pathogens, while themselves remaining unaffected. This phenomenon is attributed to the innate immune system of ticks. The focus of this project is on stimulus-induced immunoreactive peptides known as antimicrobial peptides. In chapter 2, an attempt was made to identify a homolog of the anti Gram-positive and bacteriostatic peptide microplusin, in the salivary glands of the argasid tick Ornithodoros savignyi. It was reported previously that tissue and life stage specific expression of this transcript occurs in the fat body of adult, fully fed, female Rhipicephalus (Boophilus) microplus ticks. The positive control used for this study was unsuccessful due to the incorrect tissue and life stage of R. (B.) microplus ticks. No significant homolog was identified due to the possible existence of stringent regulation of expression as well as differences in the induction stimuli between argasid and ixodid ticks. Lysozyme catalyzes the cleavage of the β-1,4 glycosidic bond between N-acetyl muramic acid and N-acetyl glucosamine of the peptidoglycan layer of bacterial cell walls affording the molecule antibacterial activity. In argasid ticks, lysozyme was observed to be induced by feeding. In chapter 3, an attempt was made to elucidate the O. savignyi homolog of the O. moubata lysozyme molecule. The partial sequence obtained revealed the presence of a lysozyme homolog in O. savignyi. The tissue expression profile revealed constitutive expression in the midgut and ovaries and induction of transcription in the hemolymph upon feeding. In salivary glands, upregulation was observed following ingestion of Gram-positive bacteria. In chapter 4, the tissue expression profile of O. savignyi defensin was investigated. It was found that transcription is induced following the ingestion of Gram-positive bacteria, while in the hemolymph upregulation was observed upon feeding. Furthermore, chapter 4 saw the attempts made at the RNAi mediated silencing of the lysozyme and defensin transcripts. Silencing, analysed by real time PCR, was not efficient as no statistically significant silencing was observed. Observation of the phenotype revealed mortality. However, statistical analysis of silencing revealed that the mortality observed was not due to silencing, but non-specific and possibly the result of injury during injection. Overall, the abovementioned experiments revealed the tissue specificity of expression of ixodid microplusin and that a more strategic approach is required for the elucidation of the argasid homolog. The partial O. savignyi lysozyme sequence was elucidated together with the tissue expression profile of this molecule and O. savignyi defensin. The RNAi experiments require optimization for future studies. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Overuse of conventional antibiotics has led to increased multidrug resistant micro-organisms. Therefore, development of alternative drugs with new mechanisms of action in the control of resistant micro-organisms is urgently needed. Defensins, one of the larger groups of naturally occurring antimicrobial peptides (AMPs), found in a variety of species, may serve as templates for the development of novel therapeutic agents. The work completed in this study is based on an antimicrobial peptide (AMP), Os, derived from the C-terminus of a tick Ornithodoros savignyi defensin isoform 2 (OsDef2). OsDef2 was found to be active against Gram-positive bacteria only, whereas Os, showed bactericidal activity towards both Gram-positive and Gram-negative bacteria. In this study a series of synthetic shorter peptides, based on the sequence of Os, was utilised in order to determine whether shorter peptides would retain their antibacterial activity and selectivity. Initial screening indicated that only two fragments, Os(3-12) and Os(11-22), were active towards the tested Gram-negative and Gram-positive bacteria. The minimum bactericidal concentrations (MBCs) of the two fragments were determined and ranged from 30 μg/ml to 120 μg/ml. The MBCs of the parent peptide, Os (1.88 to 15 μg/ml), was considerably lower than that of Os(3-12) and Os(11-22). As previously observed for Os, neither of the peptides showed cytotoxic effects towards eukaryotic cells. The amidated analogue of one of the active peptides, Os(11-22)NH2, was further evaluated in terms of its secondary structure, antibacterial and antioxidant activities as well as cytotoxicity. Amidation increased the activity of Os(11-22) 16 fold towards B. subtilis (MBC of 1.88 μg/ml) and 32 fold towards both Escherichia coli and Pseudomonas aeruginosa (MBC of 3.75 μg/ml), whereas a 2 fold decrease in activity was observed against Staphylococcus aureus (MBC of 60 μg/ml). Circular dichroism data showed that amidation altered the secondary structure of Os(11-22) from α-helical to mostly random coiled. In the presence of 30% serum the activity of Os(11-22)NH2 unexpectedly increased 8 fold against S. aureus (MBC of 7.5 μg/ml), but decreased 32 fold against E. coli (MBC of 120 μg/ml). The activity of Os(11-22)NH2 in 100 mM NaCl decreased 4 fold against E. coli (MBC of 15 μg/ml), but was completely lost (MBC >120 μg/ml) against S. aureus. The kinetics of bactericidal activity indicated that Os(11-22)NH2 killed B. subtilis and E. coli within 30 min and 120 min, respectively, whereas Os killed both bacteria within 5 min. Even at high concentrations Os(Os(11-22)NH2 was non-toxic towards human erythrocytes and SC-1 cells, moreover an increase in SC-1 cell number was observed at 120 μg/ml. The peptide showed strong antioxidant activity and was found to be 4 fold more active than glutathione (GSH), however Os was 3.4 fold more antioxidative than Os(11-22)NH2. Os(11-22)NH2 can be considered a dual functional peptide, since it possesses both antibacterial and antioxidant activity. The amidated peptide has the potential for use against the damaging effects of oxidative stress associated with infectious diseases and recovery of chronic wounds. Further investigation into structure-function properties of Os(11-22)NH2 is necessary. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Biochemistry / Unrestricted

In this study we investigate the synergistic effect of low frequency ultrasonication (14, 22, and 47 kHz) on antimicrobial activity of Cecropin P1 against Escherichia coli. The hypothesis was tested by comparing three different treatments (1) ultrasonication only (2) Cecropin P1 only (3) combination of both. The results showed that the combined treatment deactivate E. coli more efficiently by six order of magnitude. The mechanism of membrane permeabilization due to Cecropin P1 is also investigated using dye leakage experiment. The result indicated pore formation and carpet mechanism. Finally, a mathematical modeling is proposed to explain the synergistic effect, allowing us to make better prediction for cell deactivation.

No description available.

NKT cells are an important part of human immune system and recognize a specific set of antigens called glycolipids. Only a handful of "natural" NKT cell antigens are known till date. Although NKT cells play a protective role against pathogenic organisms, imbalances in NKT cell functions are implicated in many diseases including asthma. Allergic asthma, a Th2 driven inflammation of airways, is primarily caused by inhalation of environmental allergens. In the last decade, inhaled allergen Aspergillus fumigatus has been under scrutiny for the presence of NKT cell antigens that might trigger asthma. We successfully isolated, characterized and synthesized a "natural" antigenic glycolipid which activates NKT cells in CD1d dependent manner. When this glycolipid is administered intranasally to mice, WT but not CD1d-/- mice developed airway hyperreactivity (AHR), which is a cardinal feature of asthma. Our results indicate that this glycolipid also triggers the production of key cytokines responsible for development of airway hyperreactivity, namely IL-4 and IL-13. Widespread use of antibiotics has convoluted the problem of antimicrobial resistance. Our research group has developed a novel class of antimicrobial peptide mimics called Ceragennins. These cholic acid based antimicrobial compounds have many desirable properties including low MICs, effectiveness against biofilms, and relatively low manufacturing cost. In order to advance the clinical development of Ceragennins, we developed analytical methods for qualitative and quantitative determination of these compounds in complex biological matrices. These methods were also used for carrying out the stability studies of Ceragenins under varying pH and temperatures

The recent upsurge of multidrug resistant bacteria (MDRB) among global communities has become one of the most serious challenges facing health professionals and the human population worldwide. Cationic ultrashort antimicrobial peptides (USAMPs) are a promising group of molecules that meet the required criteria of novel antimicrobial drug development. UP-5, a novel penta-peptide, displayed significant antimicrobial activities against various standard and clinical isolates of MDRB. UP-5 displayed MICs values within the range of (10–15 M) and (55–65 M) against Gram-positive and Gram-negative bacteria, respectively. Furthermore, UP-5 displayed antibiofilm activity with minimum biofilm eradication concentration (MBEC) value as equal to twofold higher than MIC value. At the same inhibitory concentrations, UP-5 exhibited very low or negligible toxicity toward human erythrocytes and mammalian cells. Combining UP-5 with conventional antibiotics led to a synergistic or additive mode of action that resulted in the reduction of the MIC values for some of the antibiotics by 99.7% along a significant drop in MIC values of the peptide. The stability profile of UP-5 was evaluated in full mouse plasma and serum with results indicating a more stable pattern in plasma. The present study indicates that USAMPs are promising antimicrobial agents that can avoid the negative characteristics of conventional antimicrobial peptides. Additionally, USAMPs exhibit good to moderate activity against MDRB, negligible toxicity, and synergistic outcomes in combination with conventional antimicrobial agents.

No description available.

The objective of this dissertation is to demonstrate different applications of ssNMR, with particular emphasis on uses in polymeric and biosciences. First, dynamics investigations on two polymers will be discussed: (1) disulfonated poly(arylene ether sulfone)s /poly(ethylene glycol) blends (BPS-20_PEG), which are under development as chlorine-resistant reverse osmosis (RO) membrane alternatives to aromatic polyamide (PA) technology, and (2) poly(arylene ether sulfone)s modified with 1,4-cyclohexyl ring units to improve processability. Simple cross-polarization magic-angle-spinning (CPMAS) experiments compared the chlorine tolerance of BPS-20_PEG and PA. Techniques capable of detecting motional geometries and rates on timescales from nanoseconds to seconds, including relaxation time measurements, were applied. Correlations were established between relaxation time and water permeability for the RO membranes, and between relaxation time and polydispersity in the 1,4- cyclohexyl ring modified polymer. Next, 31P and 2H static ssNMR experiments evidencing the formation of toroidal pores and thinned bilayers in oriented zwitterionic and anionic phospholipid bilayers, (biomembrane mimetic systems), by the antimicrobial peptides (AMPs) magainin-2 and aurein-3.3, will be mentioned. The toroidal pore geometries induced by magainin-2 were different than those produced by aurien-3.3. The most prominent features were observed in 2H spectra, implying greater interaction of the peptides with hydrophobic lipid acyl chains. Following this, a new two-dimensional homonuclear dipolar recoupling MAS experiment, capable of correlating long range 13C-13C spin pairs in a uniformly/ extensively 13C-labeled biomolecule, will be introduced. This technique was demonstrated on 13C-labeled versions of Glutamine and Glycine-Alanine-Leucine. Experiments involving the recoupling of all 13C-13C spin pairs, and experiments with selective recoupling using Gaussian or cosine-modulated Gaussian pulses, were demonstrated. Finally, work using static 1H- 13C CP ssNMR to selectively detect interfacial water around hydrophobic C60 will be recounted. This project exploited the distance limitation of CP, and 1H spin-lattice relaxation times, to separate the influence of bulk and interfacial water on the spectra. Results indicated that the tumbling of interfacial water is slowed by a factor of 105 compared to bulk water, providing it with a solid-like character, and allowing the hydration shell to be stable at temperatures above the freezing point of water. / Ph. D.

Dermicidina (DCD) é um gene mapeado no cromossomo 12, lócus 12q13.1, e codifica uma proteína de 110 aminoácidos, que sofre um processamento proteolítico, gerando peptídeos ativos. O peptídeo C-terminal (DCD-1L) de 48 aminoácidos tem uma carga -2, e exerce função antibacteriana e antifúngica, e o peptídeo C-terminal splice variante, denominado DCD-SV de 59 aminoácidos, tem carga neutra, e suas propriedades ainda não foram estabelecidas. Neste trabalho são apresentados os resultados da expressão, purificação e sequenciamento da DCD nativa produzida em E. coli BL21 transformada com o vetor pAE-DCD. Na segunda parte são descritas as análises físico-químicas e bioquímicas da interação dos peptídeos sintéticos DCD-1L e DCD-SV com vesículas lipídicas gigantes e vesículas unilamelar grandes sintetizadas com palmitoil-oleoil-fosfatidilcolina. As preferenciais estruturais dos peptídeos foram investigadas por espectroscopia de Dicroísmo Circular. Nossos resultados sugerem que a DCD-SV tem alta propensão para adotar uma estrutura helicoidal permitindo sua inserção e oligomerização em membranas biomiméticas, e possível formação de canais de condutância molecular. / Dermicidin (DCD) is mapped a gene on chromosome 12, locus 12q1.13 whose 110 amino acids protein is proteolytically processed to N and C-terminal peptides. The 48-amino acid C-terminal peptide (DCD-1L) has -2 net charges and display antibacterial and antifungal properties and the 59-amino acid splice variant C-terminal peptide (DCD-SV) has neutral net charge; however, its structure and biological function are unknown. Here we show the results of expression, purification and amino acid sequencing of recombinant DCD protein produced in E.coli transformed with pAE-DCD vector. We also describe the results of physical-chemical and biochemical analyses showing the visible differences between the interactions of DCD-1LL and DCD-SV synthetic peptides with giant unilamellar vesicles and large unilamellar vesciles made of palmitoyl-oleoyl phosphatidylcholine, used as biomimetic membranes. The structural preferences of peptides were analyzed by circular dichroism spectroscopy. Our results suggest that DCD-SV peptide has higher propensity to adopt helicoidal structure enabling it to insert into mimetic membranes, undergo oligomerization and formation of conductance channel.

Peptídeos antimicrobianos são vistos como alternativas promissoras a serem empregadas pela iindústria farmacêutica no controle de infecções causadas por microrganismos, como também na indústria alimentícia, onde podem desempenhar papéis como conservantes naturais de alimentos. Plantaricina149 é um membro deste grupo, sendo composto por 22 resíduos de aminoácidos, com natureza catiônica e atividade inibitória sobre algumas bactérias patogênicas. Neste trabalho, foram sintetizados diferentes peptídeos análogos à Plantaricina149 para investigar suas ações sobre microrganismos (bactérias e fungos), a fim de correlacionar estes estudos com a ação lítica do peptídeo em modelos de membrana diversos (monocamadas e vesículas fosfolipídicas). A interação de Plantaricina149 com estes sistemas foi monitorada pelas espectroscopias de dicroísmo circular e fluorescência, ensaios de tensão superficial, calorimetria e ressonância plasmônica de superfície, e mostrou ser altamente específica para superfícies fosfolipídicas que apresentam densidade de cargas negativas, tais como a membrana celular de bactérias. A interação eletrostática inicial que se estabelece entre o peptídeo e os fosfolipídios é de extrema importância, sendo capaz de induzir uma estruturação helicoidal na região C-terminal do peptídeo, enquanto a região Nterminal contribui com as interações hidrofóbicas necessárias para a penetração do peptídeo nas camadas fosfolipídicas levando a ruptura das mesmas. De forma semelhante, a atividade antimicrobiana de Plantaricina149a (e alguns de seus análogos) também mostrou ser resultado das interações das duas regiões da molécula, e foi afetada com a retirada ou modificação da região N-terminal do peptídeo. Com a deleção desta região, o peptídeo passou a ter somente ação bacteriostática sobre Staphylococcus aureus e Pseudomonas aeruginosa, perdendo a capacidade bactericida. / Antimicrobial peptides are seen as promising alternatives to be employed in pharmaceutical industry for controlling infections caused by microorganisms, and also in food industry, where they can play roles as natural food preservatives. Plantaricina149 is a member of this group, constituted of 22 amino acid residues, cationic in nature and presenting inhibitory activity against some pathogenic bacteria. In this work, different Plantaricina149 analog peptides were synthesized to investigate their action against microorganisms (bacteria and fungi), with the aim of correlating these studies with the lytic action of the peptide on several membrane models (phospholipid monolayers and vesicles). The Plantaricina149 interaction with these systems was monitored by circular dichroism and fluorescence spectroscopies, surface tension assays, calorimetry and surface plasmon resonance, and showed to be highly specific to phospholipid surfaces that present negative charge density, such as the bacteria cell membrane. The initial peptide-phospholipids electrostatic interaction is extremely important, and it is capable of inducing a helical structure in the peptide C-terminal region, while the Nterminal region contributes with the hydrophobic interactions needed to the peptide penetration in the phospholipid layers and to the disruption of them. Similarly, the Plantaricina149 antimicrobial activity has also proved to be a result of the interactions from the two regions of the molecule, and it was strongly affected by the removal or modification of the peptide N-terminal region. Promoting the deletion of this region has left the peptide only with a bacteriostatic action against Staphylococcus aureus and Pseudomonas aeruginosa, removing its bactericide ability.