RESUMO
Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.
Assuntos
Apolipoproteínas/química , Membrana Celular/química , Proteínas de Insetos/química , Legionella/química , Mariposas/microbiologia , Animais , Apolipoproteínas/isolamento & purificação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colina/farmacologia , Corantes Fluorescentes/química , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Insetos/isolamento & purificação , Legionella/efeitos dos fármacos , Legionella/crescimento & desenvolvimento , Legionella/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Mariposas/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Ligação Proteica , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Phosphatidycholine (PC) is the major membrane-forming phospholipid in eukaryotes but it has been found in only a limited number of prokaryotes. Bacteria synthesize PC via the phospholipid N-methylation pathway (Pmt) or via the phosphatidylcholine synthase pathway (Pcs) or both. Here, we demonstrated that Legionella dumoffii has the ability to utilize exogenous choline for phosphatidylcholine (PC) synthesis when bacteria grow in the presence of choline. The Pcs seems to be a primary pathway for synthesis of this phospholipid in L. dumoffii. Structurally different PC species were distributed in the outer and inner membranes. As shown by the LC/ESI-MS analyses, PC15:0/15:0, PC16:0/15:0, and PC17:0/17:1 were identified in the outer membrane and PC14:0/16:0, PC16:0/17:1, and PC20:0/15:0 in the inner membrane. L. dumoffii pcsA gene encoding phosphatidylcholine synthase revealed the highest sequence identity to pcsA of L. bozemanae (82%) and L. longbeachae (81%) and lower identity to pcsA of L. drancourtii (78%) and L. pneumophila (71%). The level of TNF-α in THP1-differentiated cells induced by live and temperature-killed L. dumoffii cultured on a medium supplemented with choline was assessed. Live L. dumoffii bacteria cultured on the choline-supplemented medium induced TNF-α three-fold less efficiently than cells grown on the non-supplemented medium. There is an evident effect of PC modification, which impairs the macrophage inflammatory response.