However, when compared with magainin-2, a typical α-helical AMP with potent lytic activity [30], the lytic properties of cementoin, elafin or pre-elafin/trappin-2 toward P. aeruginosa and artificial membranes are very weak. We have also tested the ability SHP099 clinical trial of pre-elafin/trappin-2 and its domains to interfere with the expression of known P. aeruginosa virulence factors and compared this activity to that of azithromycin, an antibiotic that perturbs cell to cell communication

in P. aeruginosa and significantly retards biofilm formation [31, 32]. Pre-elafin/trappin-2 and elafin, but not cementoin, were found to reduce biofilm development and the secretion of pyoverdine and this correlated with the ability of these peptides to bind DNA in vitro and to accumulate within the bacterial cytosol. Rather than causing extensive cell lysis, PD0325901 datasheet our data thus suggest that pre-elafin/trappin-2

and elafin attenuate the expression of some P. aeruginosa virulence factors, possibly through acting on an intracellular target. Results The cementoin domain of pre-elafin/trappin-2 adopts an α-helical conformation in the presence of membrane mimetics Different experiments were performed to characterize the structure of cementoin and its interaction with membranes. First, we recorded circular dichroism (CD) spectra in the presence or absence of trifluoroethanol (TFE), which mimics a membrane environment [33] (Fig. 1A). In an aqueous solution, the CD spectrum is typical

of an unstructured protein with a prominent negative peak at 199 nm. When TFE was added, the intensity of this peak decreased concomitantly with the Doramapimod molecular weight appearance of minima around 205 nm Mannose-binding protein-associated serine protease and 222 nm whose intensity increased with the concentration of TFE. This is characteristic of an α-helical structure and the α-helical content of cementoin was estimated to be 48% in 50% TFE and up to 58% in 75% TFE. The observed isodichroic point at 203 nm indicates that the transition between the unstructured to the α-helical conformation is a two-state transition. Hence, a hydrophobic environment either induces or stabilizes α-helices in cementoin. This is in agreement with the AGADIR algorithm (Fig. 1D), which predicts the formation of two α-helices in cementoin: helix 1 with residues 10- 16 and helix 2 with residues 24-31, for a predicted total α-helical content of 39%. Figure 1 Biophysical characterization of cementoin. A) CD spectra of cementoin with varying concentrations of TFE (up to 75%). The vertical lines indicate 208 and 222 nm, i.e. characteristic wavelengths for assessing the presence of α-helices. B) 2 D 15N-HSQC spectrum of cementoin in the presence of 50% TFE. Backbone assignments are shown. Side-chain Asn, Gln and Arg doublets are depicted with a line between the two resonances while unassigned additional peaks (potentially arising from slow exchange, see text) are labeled by an asterisk (*). C) SSP analysis of backbone Cα and Cβ chemical shifts.