design of selective peptide antibiotics by using the sequence moment concept

New antibiotics against multidrug-resistant bacteria are urgently needed, but rapid acquisition of resistance limits their usefulness. Endogenous antimicrobial peptides (AMPs) with moderate selectivity, but multimodal mechanism of action, have remained effective against bacteria for millions of years. Their therapeutic application, however, requires optimizing the balance between antibacterial activity and selectivity, so that rational design methods for increasing selectivity are highly desirable. We have created training (n=36) and testing (n=37) sets from frog-derived AMPs with determined therapeutic index (TI). The 'sequence moments' concept then enabled us to find a one-parameter linear model resulting in a good correlation between measured and predicted TI (r2=0.83 and 0.64 for each set, respectively). The concept was then used in the AMP-Designer algorithm to propose primary structures for highly selective AMPs against Gram-negative bacteria. Testing the activity of one such peptide produced a TI>200 as compared to the best AMP in the data-base, with TI=125

Effects on APC antigen presenting cells of short-term interaction with the human human host defense peptide beta-defensin 2

International audiencebeta-Defensins are antimicrobial peptides that exert their host-defense functions at the interface between the host and microbial biota. They display a direct, salt and medium sensitive cidal activity, in vitro, against a broad spectrum of bacteria and fungi and there is increasing evidence that they also play a role in alerting and enhancing cellular components of innate and adaptive immunity. Their interaction with biological membranes plays a central role in both these types of activities. In this study we have investigated the interaction of fluorescently labelled human beta defensin 2 (hBD2) with monocytes, macrophages and immature dendritic cells, observing a differential capacity to be rapidly internalised into these cells. Complementary microscopy techniques (TEM, optical and infrared) were used to explore the functional and biological implications of these interactions on immature dendritic cells. Short-term exposure to the peptide resulted in significant alterations in membrane composition and re-organization of the endomembrane system, with induction of degranulation. These events may be associated with the antigen-presenting activities or the chemotaxis of iDC, which appears to occur via both CCR6-dependent and independent mechanisms

Primate cathelicidin orthologues display different structures and membrane interactions

The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host-cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene (encoding LL-37) in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the helical conformation and tendency to aggregate in solution of the peptide. In the present study, we have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37 and orang-utan LL-37, using several complementary biophysical and biochemical methods.CD and ATR (attenuated total reflection)-FTIR (Fourier-transform IR) spectroscopy on model membranes indicate that RL-37, which is monomeric and unstructured in bulk solution [F-form (free form)], and human LL-37, which is partly structured and probably aggregated [A-form (aggregated form)], bind biological membranes in different manners. RL-37 may insert more deeply into the lipid bilayer than LL-37, which remains aggregated. AFM(atomic forcemicroscopy) performed on the same supported bilayer as used for ATR-FTIR measurements suggests a carpetlike mode of permeabilization for RL37 and formation of more defined worm-holes for LL-37. Comparison of data from the biological activity on bacterial cells with permeabilization of model membranes indicates that the structure/aggregation state also affects the trajectory of the peptides from bulk solution through the outer cell-wall layers to the membrane. The results of the present study suggest that F-form cathelicidin orthologues may have evolved to have primarily a direct antimicrobial defensive capacity, whereas the A-forms have somewhat sacrificed this to gain host-cell modulating functions

Structuring and interactions of human beta-defensins 2 and 3 with model membranes

\u3b2-Defensins play an important role in both innate and adaptive immunity. Interaction with biological membranes appears to be a central theme in modulating these activities, leading to different consequences such as membrane lysis, translocation into the cytoplasm or transfer to a receptor. We have investigated the structuring of human \u3b2-defensins (hBD2 and hBD3) and rationally designed variants, in relation to their interactions with real and model membranes. Our results indicate that structural features, such as the helical N-terminal domains and oligomerisation at the membrane surface, may modulate the efficiency of membrane insertion and selectivity for microbial or host-cell membranes. We propose that both peptides interact with membranes as extended \u3b2-sheet platforms that present amphipathic helices for insertion into the lipid bilayer

Evolution of the beta defensin 2 gene in primates

With the aim of further investigating the molecular evolution of beta defensin genes, after having analysed beta defensin 1 (DEFB1) in humans and several nonhuman primate species, we have studied the evolution of the beta defensin 2 gene (DEFB2), which codifies for a peptide with antimicrobial and chemoattractant activity, in humans and 16 primate species. We have found evidence of positive selection during the evolution of orthologous DEFB2 genes at two points on a phylogenetic tree relating these primates: during the divergence of the platyrrhines from the catarrhines and during the divergence of the Cercopithecidae from the Hylobatidae, Great Apes and humans. Furthermore, amino acid variations in Old World Monkeys Seem to centre either on residues that are involved in oligomerisation in the human molecule, or that are conserved (40-80%) In beta-defensins in general. It is thus likely that these variations affect the biological function of the molecules and suggest that their synthesis and functional analysis might reveal interesting new information as to their role in innate immunity. \ua9 2003 Nature Publishing Group All rights reserved