Study of the interaction of an antimicrobial protein (StAsp-PSI) with phospholipid bilayer membranes

MUÑOZ, FERNANDO; PALOMARES-JERÉZ, MARÍA FRANCISCA; DALEO, GUSTAVO; GUEVARA, MARÍA GABRIELA; VILLALAÍN, JOSÉ

Conferencia; International Conference on Antimicrobial Research (ICAR 2010); 2010

Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of a saposin-like domain. Hence, PSI is called a swaposin domain. Previously, we have reported the cloning, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI is cytotoxic to human pathogens in a dose-dependent manner, but this effect was not observed on human red blood cells at all concentrations assayed. The StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell membrane, leading to increased permeability and lysis. However, the mechanism by which StAsp-PSI interacts with the plasma membrane is not well understood. The aim of this work was to analyze the properties of StAsp-PSI/lipid interactions. The results obtained show that StAsp-PSI was able to cause surface destabilization and leakage in a dose-dependent manner in membrane vesicles composed of EPC/EPA (5:4) and EPC/EPG (5:4). A decrease in the StAsp-PSI leakage ability was detected when vesicles were composed of zwitterionic lipids (EPC and EPC/TPE (5:2)) or a lipid extract from bovine liver. Additionally, the presence of cholesterol in the vesicle composition decreased the ability of StAsp-PSI to cause leakage in a dose-dependent manner. In order to analyze StAsp-PSI conformational changes, circular dichroism (CD) assays were performed. Data obtained show that StAsp-PSI helical conformation was reduced by 50, 70 and 20 % after incubation of this protein with membranes composed of EPC/EPA (5:2), EPC/EPG (5:2), and EPC/Chol (5:2)  respectively. Results obtained from differential scanning calorimetry (DSC) showed that StAsp-PSI was able to significantly perturb anionic bilayers (composed of dimyristoylphosphatidyl glycerol, DMPG and dimyristoylphosphatidyl serine, DMPS).  However, there was a lower effect of StAsp-PSI on zwitterionic bilayers (dimyristoylphosphatidylcholine, DMPC). Infrared spectroscopy (IR) spectra results show that minor changes were detected in the StAsp-PSI Amide I´ region in the presence of anionic or neutral membranes. Therefore, the results obtained in this work suggest  that: a) the content of high amounts of anionic lipids in the plasma membrane of prokaryotic cells, b) the decrease observed in the StAsp-PSI vesicle leakage ability in the presence of cholesterol, and c) the fact that, at lower amounts of StAsp-PSI, vesicle leakage is only detected in negative vesicles, could explain the differences observed in the ability of StAsp-PSI to kill human bacterial pathogens (E. coli, S. aureus, and B. cereus) but not human cells (lymphocytes and erythrocytes).