Publicação
Study of the mechanism of action of the antimicrobial peptide rBPI21 in gram-negative bacteria
| Resumo: | Antimicrobial peptides (AMPs) are currently looked as new candidates to overcome the bacterial resistance against therapeutic antibiotics. However, their mechanism of action remains unclear, despite several theories having been proposed. The understanding of the processes that govern AMPs activity is the best way to provide rational design of new antibiotics for further clinical use against bacterial infections. Human AMPs are part of the innate immune system and synthetic versions of these AMPs are good candidates, due to their low toxicity and high antimicrobial activity. It is believed that the mode of action of these AMPs involves their action at the membrane level. This thesis is focused on the study of the interaction of a fragment based on the N-terminal region of a human antimicrobial protein, the bactericidal/permeability increasing protein (BPI), with biomembrane model systems and bacterial cells. The fragment, named rBPI21, has antimicrobial properties and neutralizes the effect of the lipopolysaccharide (LPS) during bacterial infections. The thesis describes the use of biophysical strategies in order to unravel the fundamental steps involved in the bactericidal activity of rBPI21. Membrane selectivity was quantified using a range of biophysical techniques and reported in Chapters III and V. Chapter III reveals that rBPI21 prefers negatively charged liposome systems containing phosphatidylglycerol, which mimic bacterial membranes. The preference for the anionic phosphatidylglycerol membranes is followed by membrane aggregation/fusion and, at higher rBPI21 concentrations, there is a leakage of liposome content, as described in Chapter IV. Liposomes fusion leads to multilamellar membrane structures, as studied in Chapter V by small angle X-ray scattering. Previous studies revealed that rBPI21 was able to induce membrane perturbations on negatively charged membrane model systems mimicking the membranes of bacteria. On the other hand, membrane model systems that mimic eukaryotic membranes remain unaffected by the presence of rBPI21. In Chapter VI, the interaction of rBPI21 with bacteria was studied using atomic force microscopy, and the results were correlated with those obtained with membrane model systems in order to unravel the possible mechanism of action of the peptide. rBPI21 was shown to induce membrane perturbations, culminating in bacterial cells content leakage, both on the Gram-negative bacteria Escherichia coli and on the Gram-positive Staphylococcus aureus. The interaction of rBPI21 with bacteria was decreased in the presence of free lipopolysaccharide aggregates, demonstrating the affinity of rBPI21 for free LPS, as studied by force spectroscopy. The overall observed results potentiate the use of the rBPI21 in clinics against bacterial infections. Also, the development of new synthetic peptides based on rBPI21 structure is a valuable route to develop new therapeutic agents with antibacterial properties. |
|---|---|
| Autores principais: | Domingues, Marco André Manso, 1985- |
| Assunto: | Membranes Teses de doutoramento - 2012 Peptides Anti-bacterial agents Bacteria |
| Ano: | 2012 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Antimicrobial peptides (AMPs) are currently looked as new candidates to overcome the bacterial resistance against therapeutic antibiotics. However, their mechanism of action remains unclear, despite several theories having been proposed. The understanding of the processes that govern AMPs activity is the best way to provide rational design of new antibiotics for further clinical use against bacterial infections. Human AMPs are part of the innate immune system and synthetic versions of these AMPs are good candidates, due to their low toxicity and high antimicrobial activity. It is believed that the mode of action of these AMPs involves their action at the membrane level. This thesis is focused on the study of the interaction of a fragment based on the N-terminal region of a human antimicrobial protein, the bactericidal/permeability increasing protein (BPI), with biomembrane model systems and bacterial cells. The fragment, named rBPI21, has antimicrobial properties and neutralizes the effect of the lipopolysaccharide (LPS) during bacterial infections. The thesis describes the use of biophysical strategies in order to unravel the fundamental steps involved in the bactericidal activity of rBPI21. Membrane selectivity was quantified using a range of biophysical techniques and reported in Chapters III and V. Chapter III reveals that rBPI21 prefers negatively charged liposome systems containing phosphatidylglycerol, which mimic bacterial membranes. The preference for the anionic phosphatidylglycerol membranes is followed by membrane aggregation/fusion and, at higher rBPI21 concentrations, there is a leakage of liposome content, as described in Chapter IV. Liposomes fusion leads to multilamellar membrane structures, as studied in Chapter V by small angle X-ray scattering. Previous studies revealed that rBPI21 was able to induce membrane perturbations on negatively charged membrane model systems mimicking the membranes of bacteria. On the other hand, membrane model systems that mimic eukaryotic membranes remain unaffected by the presence of rBPI21. In Chapter VI, the interaction of rBPI21 with bacteria was studied using atomic force microscopy, and the results were correlated with those obtained with membrane model systems in order to unravel the possible mechanism of action of the peptide. rBPI21 was shown to induce membrane perturbations, culminating in bacterial cells content leakage, both on the Gram-negative bacteria Escherichia coli and on the Gram-positive Staphylococcus aureus. The interaction of rBPI21 with bacteria was decreased in the presence of free lipopolysaccharide aggregates, demonstrating the affinity of rBPI21 for free LPS, as studied by force spectroscopy. The overall observed results potentiate the use of the rBPI21 in clinics against bacterial infections. Also, the development of new synthetic peptides based on rBPI21 structure is a valuable route to develop new therapeutic agents with antibacterial properties. |
|---|