School of Chemistry - Theses

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    Study of the membrane interactions of β – amyloid (Aβ) and cytolytic peptides
    Jamasbi, Elahehsadat ( 2016)
    Analogues of the membrane-active peptides, melittin and the amyloid beta peptide (Aβ), were synthesized and studied in model membrane and live cells. Melittin (MLT) is a peptide from bee venom that lyses cell membranes and amyloid Abeta (Aβ) is a hallmark of Alzheimer’s disease. The interaction of MLT and Aβ peptides with model mebranes and live cells were investigated. The biophysical, cell binding and toxicity properties of MLT and Aβ peptides were studied using CD spectroscopy, fluorescence, light and confocal microscopy. Melittin is a lytic peptide with a broad spectrum of activity against both eukaryotic and prokaryotic cells. To understand the role of proline and the thiol group of cysteine in the cytolytic activity of MLT, native MLT and cysteine-containing analogues were prepared using solid phase peptide synthesis. The antimicrobial and cytolytic activities of the monomeric and dimeric MLT peptides against different cells and model membranes were investigated. The results indicated that the proline residue was necessary for antimicrobial activity and cytotoxicity and its absence significantly reduced lysis of model membranes. Although lytic activity against model membranes decreased for the MLT dimer, haemolytic activity was increased. The native peptide and the MLT-P14C monomer were mainly unstructured in buffer while the dimer adopted a helical conformation. In the presence of neutral and negatively charged vesicles, the helical content of the three peptides was significantly increased. The lytic activity, therefore, is not correlated to the secondary structure of the peptides and, more particularly, on the propensity to adopt helical conformation. The mechanism of membrane disruption by MLT of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labelling with maleimide-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labelled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, haemolysis and cytotoxicity, but not essential for cell membrane disruption. The Aβ peptides found in plaques in the brain has been widely recognised as a hallmark of Alzheimer’s disease although the underlying mechanism is still unknown. Aβ40 and Aβ42 peptides were synthesized and the effects of mutation and phosphorylation of the Aβ peptide on neuronal cells were investigated. The A2T mutation appeared to reduce cytotoxicity and lessen binding of Aβ40 peptides to neuron cells while phosphorylation at Ser8 increased cell binding and β-sheet formation of Aβ42 peptides. Confocal microscope imaging of live cells indicated differences in behaviour between mutant (A2T) and wild type Aβ40 and phosphorylated Aβ42. Microscopy techniques for studying the interaction of melittin and Aβpeptides with model membrane provided insight into peptide interactions with live cells. The aim of this thesis was to study the interaction of membrane-active peptides with cells and model membranes, in particular, membrane lytic and amyloid peptides. The role of key residues and the effect of membrane lipid composition and cell lines on peptide function was determined.
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    Nanomechanical studies of the interaction of antimicrobial peptides with bacterial cells
    Mularski, Anna ( 2016)
    Antimicrobial peptides (AMPs) are promising therapeutic alternatives to conventional antibiotics. Many AMPs are membrane-active but their mode of action in killing bacteria or in inhibiting their growth remains elusive. Recent studies indicate the mechanism of action depends on peptide structure and lipid components of the bacterial cell membrane. To date, most of these studies have been conducted with synthetic membrane systems, which neglect the possible role of bacterial surface structures in these interactions. Here, we use atomic force microscopy (AFM) to study the interactions of three different AMPs on living Klebsiella pneumoniae bacterial cells. K. pneumoniae are recognized as a serious cause of nosocomial infections and a source of shared antimicrobial resistance. Typically, K. pneumoniae assemble a polysaccharide layer (known as the capsule) on the outside of the cell envelope, taking the form of a hydrated polyelectrolyte network that can grow up to several hundred nanometers thick, and can provide protection from environmental stresses, including desiccation, detergents, antibiotics and host immune defenses. It also plays a role in bacterial adherence to surfaces (such as medical devices) through the formation of biofilms. In situ biophysical measurements were performed to understand how the antimicrobial modulate various biophysical behaviours of individual bacteria, including the turgor pressure, cell wall elasticity, and bacterial capsule thickness and organisation. Firstly, the interactions of a melittin-derived lytic peptide with live K. pneumoniae cells were studied revealing that exposure to the peptide had a significant effect on the turgor pressure and Young’s modulus of the cell. The turgor pressure increased upon peptide addition followed by a later decrease, suggesting that cell lysis occurred and pressure was lost through destruction of the cell envelope. The Young’s modulus also increased, indicating that interaction with the peptide increased the rigidity of the cell wall. The bacterial capsule appeared unaffected by exposure to the peptide and microbiological assays determined that the presence of the capsule conferred no advantage to the wild-type over capsule deficient cells when exposed to the peptide. These findings suggest that even though the long-range electrostatic attraction between the peptide and capsule may first attract the peptide to the bacterial cell, it is the drive of the peptide to associate with the membrane that dominates once the peptide approaches the bacterial outer envelope. The methodology developed was then applied to study the interaction of the polypeptide antibiotic, colistin sulfate, with both wild-type and colistin-resistant K. pneumoniae. The capsular polysaccharides of wild-type cells were rearranged after exposure to colistin, particularly at lysis inducing concentrations, where the capsule appears to unravel. Colistin resistant mutant cells did not display the same behaviour. Microbiological assays showed that the presence of the capsule confers no advantage for wild-type over the capsule deficient cells. Genetic sequencing of the colistin resistant mutant showed that it’s ten-fold resistance to colistin is conferred by a reduction in net negative charge of the lipopolysaccharide molecules embedded in the outer membrane. Negative charge reduction could hinder the initial electrostatic binding of colistin to the outer membrane and the displacement of the divalent cations that function to bridge adjacent LPS molecules throughout the capsular polysaccharide network. Retention of the cross-linking divalent cations may explain why capsule thickness measurements did not increase in the colistin-resistant strain after colistin exposure. These results contrast with those for other K. pneumoniae strains that suggest the capsule confers colistin resistance. Finally, the interaction of caerin 1.1 with K. pneumoniae cells was studied. Caerin is a peptide similar in structure and size to melittin. As with the melittin derived peptide, the capsule of K. pneumoniae AJ218 was unchanged by exposure to caerin, indicating once again that the ionic interaction of the positively charged peptide with the negatively charged capsular polysaccharide is not a critical component of AMP interaction with K. pneumoniae AJ218 cells, the presence of a capsule conferring no advantage to wild-type over capsule-deficient cells when exposed to the AMP caerin. Time-resolved AFM images revealed that the antimicrobial peptide (AMP) caerin 1.1 caused localised defects in the cell walls of lysed Klebsiella pneumoniae cells, corroborating a pore-forming mechanism of action. The defects continued to grow during the AFM experiment, in corroboration with large holes that were visualised by scanning electron microscopy. Defects in cytoplasmic membranes were visualised by cryo-EM using the same peptide concentration as in the AFM experiments. At three times the minimum inhibitory concentration of caerin, ‘pores’ were apparent in the outer membrane. The three studies presented here demonstrate that AFM measurements can be used to track changes in bacterial cells as they lyse due to peptide interaction. In all three studies, the presence capsular polysaccharides conferred no advantage to wild-type over capsule-deficient K. pneumoniae, suggesting ionic interaction between the peptides and bacteria-bound capsular polysaccharides is not a key component of the interaction with the K. pneumoniae AJ218. Finally, AFM and cryo-EM data have shown these three peptides operate via different mechanisms of action with K. pneumoniae.
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    Interactions of a lytic peptide with supported lipid bilayers investigated using surface-selective techniques
    Rapson, Andrew Cyrus ( 2015)
    The development of antimicrobial resistance, which is the ability of microorganisms to prevent cell death by antimicrobial agents (i.e. antibiotics, antivirals), is an increasing problem. Traditional antimicrobial drugs target aspects of microorganisms responsible for infectious diseases cell structure than can be altered, greatly reducing their cell-killing efficiency. To combat this, research has been performed into the application of naturally-occurring antimicrobial peptides, sourced from venoms or host-defence systems of plants and animals. These peptides display broad-spectrum cell-killing activity by directly attacking the lipid matrix of cell membranes, thereby bypassing the means through which cells develop resistance. In this study, a proline-substituted, fluorescently-labelled analogue of the antimicrobial peptide melittin (a major component of venom from the European honey bee) was designed and synthesised. The, analogue, named melittin P14K-Alexa 430 (MK14-A430), displayed twice the membrane-lytic activity of native melittin through means of a lipid vesicle dye-release assay. The interactions of MK14-A430 with lipid membranes consisting of 1,2-dipalmitoyl-sn-glycero-3-phoshatidylcholine (DPPC) were investigated using circular dichroism and surface-selective analytical techniques. The structure and orientation of MK14-A430 were studied by circular dichroism and oriented circular dichroism. In contrast to native melittin, MK14-A430 displayed high helicity under solution conditions mimicking physiological conditions. This helicity was retained in small unilamellar vesicles, except at low peptide density in ordered DPPC vesicles. In oriented lipid multibilayers, MK14-A430 inserted completely into a transmembrane orientation in fluid-phase DPPC, while only a fraction inserted in ordered-phase bilayers (up to an observed maximum of 25%). The interaction mechanism of MK14-A430 with supported lipid bilayers (SLBs) was studied using quartz crystal microbalance with dissipation monitoring. MK14-A430 initially associated with all SLBs in a surface-aligned state, after which different behaviour was observed depending on the lipid phase. Similar to the oriented circular dichroism experiments, MK14-A430 inserted completely into a transmembrane orientation in fluid-phase DPPC, while a significant fraction in a membrane surface-aligned state was observed for both ordered-phase SLBs. The predominating orientation of MK14-A430 could be reversibly changed with the lipid phase: transmembrane above the phase transition temperature and surface-aligned below. No mass removal indicative of detergent-like membrane solubilisation was observed. The location and mobility of MK14-A430 was assessed through fluorescence of the AlexaFluor 430 label by time resolved, evanescent wave-induced fluorescence spectroscopy and anisotropy. MK14-A430 penetrated the lipid headgroup structure in pure, ordered-phase DPPC membranes but was located near the headgroup-water when cholesterol was included. MK14-A430 formed lytic pores in DPPC SLBs, and an increase in pore formation with incubation time was observed through an increase in polarity and mobility of the probe. When associated with the DPPC-Chol SLB, the probe displayed polarity and mobility that indicated a population distributed near the lipid headgroup-water interface with MK14-A430 arranged predominantly in a surface-aligned state. The information from this study showed that the lytic activity MK14-A430 occurred through a pore-forming mechanism. The lipid headgroup environment experienced by the fluorescent label where MK14-A430 displayed pore information indicated that pore formation was best described by the toroidal pore model.
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    The interaction kinetics of a melittin derivative with a phospholipid membrane
    NINGSIH, ZUBAIDAH ( 2010)
    A deeper understanding about the lipid-peptide interactions contributes significantly to the development of drug delivery systems. The utilization of a model to scrutinize the lipid-peptide interactions helps to overcome the resistance of anti-microbial agents and the unselectiveness of the anti-cancer agents. Cytolytic peptides, the peptides that able to lyse various bacteria or mammal cells, become one of the anti-microbial agent and anti-cancer agent candidates to overcome those problems. A fluorescently labeled melittin derivative and 1,2 dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) were used as a model to study the lipid-peptide interaction. One of the cytolytic peptide, melittin, is an α-helical peptide which has 6 positive charge in physiological condition. Melittin is labeled with Alexa 430. Using the steady-state fluorescence spectroscopy and Fluorescence Lifetime Imaging Microscopy (FLIM), the information on the melittin microenvironment changes through the spectral characteristic and the lifetime of Alexa 430, can be monitored. DPPC SUVs behavior was observed through the Rayleigh light scattering intensity change. The data shows that the interaction between melittin with DPPC SUVs is dependent upon the lipid-peptide ratio. At lipid-peptide ratio of 100:1, or high lipid-peptide ratio, melittin is associated with the vesicle without vesicles size change. This is indicated by the increase of Alexa 430 lifetime and quantum yield with negligible light scattering change. At lipid-peptide ratios of 50:1, 40:1 and 30:1; or the medium lipid-peptide ratio, Alexa 430 lifetime and quantum yield raise significantly followed by the increase of light scattering that be sign of further melittin insertion accompanied by the vesicles fusion. The data implies the pore formation without involving melittin insertion to the hydrocarbon chain structure. At lipid-peptide ratios of 20:1 and 10:1, or the low lipid-peptide ratio, light scattering data shows a decrease in vesicle size, which is attributed to vesicles micellization by melittin. Furthermore, the different time scale of the kinetic progress parameters; the fluorescence intensity, the lifetime and the Rayleigh light scattering intensity; signify the multi steps process which takes place during the interaction. In high lipid-peptide ratio, melittin associate with the vesicles rapidly without further vesicles size changes. In medium lipid-peptide ratio, the data implies the formation of Toroidal-pore followed by slow vesicles fusion. Meanwhile, at low lipid-peptide ratio, membrane micellization occurs in a very short time indicating the existence of the Carpet model. However, the limitation of the observation time frame gives uncertainty whether Toroidal-pore and Carpet model is the most suitable model to illustrate the lipid-peptide interaction. Hence, this research proves that melittin disrupt the zwitterionic membrane through a complex mechanism depends on the lipid-peptide ratio which need further investigation.