Study of domains formation in model membranes induced by antimicrobial peptides and their interfacial action

Author(s)

Dayane dos Santos Alvares

• Document type: Doctoral Thesis
• Press: São José do Rio Preto. 2016-06-15.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto
• Defense date: 2016-05-13
• Advisor: João Ruggiero Neto

Abstract

Antimicrobial peptides act directly on the lipid matrix of the cell membrane by perturbing the lipid packing, creating elastic stresses and mass imbalance, that are relieved by the formation of pores or defects resulting in the lytic activity. Polybia MP1, or shortly MP1, with aminoacid sequence IDWKKLLDAAKQIL-NH2, extracted from the native wasp Polybia paulista is an example of these peptides. Beyond a potent antibacterial activity, it inhibits cell proliferation in prostate and bladder cancer cell cultures. This inhibitory effect is believed to be due to the simultaneous presence of aminophospholipids: phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the outer leaflet of these cell membranes. Investigations of MP1 lytic activity in giant unilamellar vesicles (GUVs) revealed that the peptide induced some dense fluorescent regions on the vesicles that were interpreted as being due to peptide/lipid aggregation or to lipid segregation. The MP1 permeabilizing activity was also strongly modulated when fractions of aminophospholipids PS and PE were present in the lipid composition of these GUVs. It was observed that the permeability induced by MP1 increased dramatically for vesicles of PC/PE/PS (7:1:2) allowing influx of up to 10 kDa molecules. This thesis investigated the ability of MP1 in disturbing lipid packing using lipid monolayers as model membranes. Three diacylated phospholipids: PC, PE and PS were used. Compression isotherms obtained with Langmuir trough in conjunction with fluorescence and Brewster angle microscopies were used to investigate preferences of the peptide for lipid phases and its effects on the phase coexistence and on the changes of the shape, size and number of solid domains. Using diacyl chains derived from myristic and palmitic acids attached to PC, PE and PS polar heads it was possible to explore the role played by head groups and effect of the acyl chain lengths on the impact of the peptide in lipid films. These experiments showed that MP1 induced changes in both the shape and the size of solid domains. These changes were dependent on the head group and on the subphase conditions: pure water and aqueous solution of NaCl. It is remarkable that in subphase conditions in which intermolecular ionic pairs among aspartics and lysines are likely to occur, water and low salt concentration, the peptide was able to co-crystalize with DPPC lipids and induce significant changes in the shapes from “triskelion” to elongated and in the sizes of the solid domains. Change in the shape and size were also observed to DMPS and DMPE, but with different mechanisms. Due to the high preference of MP1 for PS films it was observed that part of the lipid/peptide films were irreversibly lost to the subphase, at lateral pressures compatible with that of membranes, suggesting micellization of the lipid film. These evidences for changes in the lipid packing induced by the peptide were reinforced by investigating the effect of MP1 on the influx of carboxyfluorescein in GUVs of PC/PS and of PC/SM/Chol containing or not PS. In this last composition liquid ordered (Lo) domains can be observed. In the presence of domains Lo and PS MP1 induced changes in the permeability and the formation of aggregates. The pores or defects opened in the PS containing vesicles remains opened in a minute time scale. In addition, evidences for lipid segregation was obtained from differential scan calorimetry (DSC) experiments using pure DPPS and mixed POPC/DPPS lipid vesicles showing the induction of a domain rich in peptide/anionic lipid complex and another rich in zwitterionic ones and perturbation on the lipid packing of pure anionic vesicles. (AU)

• FAPESP's process: 12/08147-8 - Study of domains formation in model membranes induced by antimicrobial peptides and their action interfacial
• Grantee: Dayane dos Santos Alvares
• Support Opportunities: Scholarships in Brazil - Doctorate