LP1A

Antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. Antimicrobial peptides (AMPs) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. Their activity is determined by numerous properties such as cationic charge, amphipathicity, size, and amino acid composition. Currently, only around 10% of known AMP sequences have experimentally solved structures. To improve our understanding of the AMP structural universe we have carried out large scale ab initio 3D modeling of structurally uncharacterized AMPs that revealed similarities between predicted folds of the modeled sequences and structures of characterized AMPs. Two of the peptides whose models matched known folds are Lebocin Peptide 1A (LP1A) and Odorranain M, predicted to form β-hairpins but, interestingly, to lack the intramolecular disulfide bonds, cation-π or aromatic interactions that generally stabilize such AMP structures. Other examples include Ponericin Q42, Latarcin 4a, Kassinatuerin 1, Ceratotoxin D, and CPF-B1 peptide, which have α-helical folds, as well as mixed αβ folds of human Histatin 2 peptide and Garvicin A which are, to the best of our knowledge, the first linear αββ fold AMPs lacking intramolecular disulfide bonds. In addition to fold matches to experimentally derived structures, unique folds were also obtained, namely for Microcin M and Ipomicin. These results help in understanding the range of protein scaffolds that naturally bear antimicrobial activity and may facilitate protein design efforts towards better AMPs.

This study characterized the protein/peptide profile of venom isolated from the spider Lasiodora sp. (Mygalomorphae, Theraphosidae) found in northeastern Brazil and determined its antimicrobial activity, toxicity against human cells, and hemolytic activity. Protein concentration of the Lasiodora sp. venom was 4.53 ± 0.38 mg/mL. SDS-PAGE showed proteins with molecular masses up to 75 kDa, some of which contained disulfide bridges. RP-HPLC analysis separate at least 12 peaks that were identified by mass spectrometry as peptides U1-theraphotoxin-Lp1a (lasiotoxin-1), U1-theraphotoxin-Lp1c (lasiotoxin-3), U3-theraphotoxin-Lsp1a (LTx5), and ω-theraphotoxin-Asp3a as well as the proteins phospholipase A2 (PLA2) and hyaluronidase. The crude venom exhibited bactericidal effect against Aeromonas sp., Bacillus subtilis, and Micrococcus luteus and fungicidal effect against Candida parapsilosis and Candida albicans. In addition, the venom exerted bacteriostatic effect against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus and fungistatic effect against Candida tropicalis and Candida krusei. The minimum inhibitory (MIC), minimum bactericidal (MBC), and minimum fungicidal (MFC) concentrations ranged from 3.9 to 500 μg/mL. The Lasiodora sp. venom decreased the viability of human peripheral blood mononuclear cells (PBMCs) by 50%-90% at concentrations of 0.1, 1, 10, and 100 μg/mL, promoting apoptosis of these cells. On the other hand, the venom showed weak hemolytic activity against Mus musculus erythrocytes (EC50: 757 μg/mL). In conclusion, the Lasiodora sp. spider venom is a rich source of antimicrobial agents. Future studies will focus on identifying antimicrobial agents present in this venom and evaluating whether these agents contribute to its cytotoxic effects against PBMCs.

To understand how the lipid composition of high density lipoprotein mediates the efflux of cellular cholesterol, we have characterized the effects of variations in the lipid composition of well defined model sonicated apolipoprotein A-I (apoA-I)-containing lipoprotein (LpA-I) particle on cholesterol efflux from cultured human skin fibroblasts. LpA-I particles with varying content of phosphatidylcholine (POPC), phosphatidylinositol, sphingomyelin, cholesterol ester, and triolein were prepared by co-sonication. Association of as little as 5 mol of phosphatidylcholine with apoA-I is sufficient to transform lipid-free apoA-I into a distinct lipoprotein-like particle that is a significantly better acceptor of cellular cholesterol. Increasing the ratio of POPC/apoA-I from 5/1 to 35.5/1 in the sonicated LpA-I is associated with a significant increase in the release of cellular cholesterol. At low POPC/apoA-I ratios, native gradient gel electrophoresis of the LpA-I shows these lipoproteins to be small complexes (around 5-6 nm), with only 1 molecule of apoA-I (Lp1A-I). At a POPC/apoA-I ratio above 11/1, LpA-I form well defined complexes that contain 2 molecules of apoA-I (Lp2A-I) and range in size from 7.6 to 7.7 nm. Inclusion of sphingomyelin into an Lp1A-I further stimulates cholesterol efflux significantly. In contrast, inclusion of either sphingomyelin or phosphatidylinositol into a sonicated Lp2A-I has no effect on cholesterol efflux. Incorporation of cholesterol ester and/or triolein into an Lp2A-I particle is associated with a small reduction in cholesterol efflux to these lipoproteins. Therefore, cholesterol efflux from human fibroblasts is directly proportional to the amount and type of phospholipid in a sonicated LpA-I particle. Changes in the conformation and charge of apoA-I that result from changes in the lipid composition of a sonicated LpA-I particle appear to directly affect the ability of the lipoprotein to bind and retain cholesterol molecules. These data therefore suggest that the adsorption/desorption of cholesterol molecules to/from a sonicated LpA-I complex may be less sensitive to interfacial lipid-lipid interactions, but may depend on a conformation-dependent ability of apoA-I to bind cholesterol.