sodium-dodecyl-sulfate and lipoteichoic-acid

The development of biomaterials integrating antimicrobial peptides (AMPs) for improved pathogen detection or use as therapeutic agents requires an understanding of how a peptide may behave once immobilized. Here, we use a combination of circular dichroism and capture assays to assess the structure-function relationship of the cationic amphipathic AMP, cecropin A (cecA), upon interaction with Gram-positive lipoteichoic acids (LTAs). In solution, cecA peptides underwent a change from a largely unstructured conformation in water to structures with significant α-helical content in the presence of both Bacillus subtilis and Staphylococcus aureus LTAs. After surface immobilization, cecA peptides attached by either C- or N-terminus were able to capture both LTAs as well as to undergo conformational changes in the presence of SDS similar to those observed in solution. However, in spite of demonstrated LTA binding activity and the ability to undergo conformational changes (i.e., with SDS), no structural changes were observed when cecA immobilized by its N-terminus was treated with either LTA preparation. On the other hand, cecA immobilized by its C-terminus underwent a conformational change in the presence of S. aureus, but not B. subtilis, LTA. These results indicate that after immobilization recognition of different targets by cationic AMPs may occur by mechanisms quite different from those in solution and that selectivity of these mechanisms is further dependent on the orientation of the immobilized peptide.

Topics: Antimicrobial Cationic Peptides; Circular Dichroism; Lipopolysaccharides; Peptides; Sodium Dodecyl Sulfate; Surface Properties; Teichoic Acids

Phenoloxidase (PO) activity is a major component of the innate immune response in arthropods. In this study, we characterized PO activity from the hair crab Erimacrus isenbeckii, which inhabits very cold regions (2.4-3.4°C) of the Bering Sea. Hemocyte lysate supernatant (HLS) prepared from E. isenbeckii was inactive HLS until activated by nonspecific agents such as sodium dodecyl sulfate and trypsin, and elicitors such as lipopolysaccharide and lipoteichoic acid from the cell wall constituent of bacteria. The PO activity was maximal at 4°C, decreased slightly at temperatures up to 60°C, and fell rapidly at 80°C. Both L-DOPA and catechol were efficient substrates for the PO (EC 1.10.3.1), with K(m) values of 0.96 and 1.15mM, respectively, whereas tyrosine and hydroquinone were not. We isolated a protein fraction from HLS as a hexamer of 75kDa units with 216.7-fold higher PO activity than that of the HLS. The N-terminal amino acid analysis of an isolated protein revealed 80% sequence identity to hemocyanins from other crabs. These results suggest that cold-adapted hemocyanin-derived PO activity is important to the survival of these crabs. This is the first report of a crab PO activity with broad temperature stability extending into the cold environment.

Topics: Animals; Brachyura; Catechols; Cell Fractionation; Cold Temperature; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Precursors; Enzyme Stability; Hemocyanins; Hemocytes; Hot Temperature; Kinetics; Levodopa; Lipopolysaccharides; Male; Monophenol Monooxygenase; Sequence Analysis, Protein; Sequence Homology; Sodium Dodecyl Sulfate; Substrate Specificity; Teichoic Acids; Trypsin

Treatment of Gram-negative bacterial infections with antimicrobial agents can cause release of the endotoxin lipopolysaccharide (LPS), the potent initiator of sepsis, which is the major cause of mortality in intensive care units worldwide. Structural information on peptides bound to LPS can lead to the development of more effective endotoxin neutralizers. Short linear antimicrobial and endotoxin-neutralizing peptide LF11, based on the human lactoferrin, binds to LPS, inducing a peptide fold with a "T-shaped" arrangement of a hydrophobic core and two clusters of basic residues that match the distance between the two phosphate groups of LPS. Side chain arrangement of LF11 bound to LPS extends the previously proposed LPS binding pattern, emphasizing the importance of both electrostatic and hydrophobic interactions in a defined geometric arrangement. In anionic micelles, the LF11 forms amphipathic conformation with a smaller hydrophobic core than in LPS, whereas in zwitterionic micelles, the structure is even less defined. Protection of tryptophan fluorescence quenching in the order SDS>LPS>DPC and hydrogen exchange protection indicates the decreasing extent of insertion of the N terminus and potential role of peptide plasticity in differentiation between bacterial and eukaryotic membranes.

Topics: Acrylamide; Amino Acid Motifs; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Differentiation; Dose-Response Relationship, Drug; Endotoxins; Humans; Lactoferrin; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Micelles; Models, Chemical; Models, Molecular; Peptides; Phosphates; Protein Binding; Protein Conformation; Protein Folding; Protein Structure, Tertiary; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence; Static Electricity; Teichoic Acids; Tryptophan

The oral organism Corynebacterium matruchotii was investigated for the presence of lipoteichoic acid, as this common polyanionic macroamphiphilic component of Gram-positive bacteria has been implicated in phenomena related to calcium binding. Phenol-water extraction followed by a small-scale, hydrophobic-interaction chromatography step yielded carbohydrate-containing preparations that were distinguished from lipoteichoic acid by their low phosphorus content. Subsequently, large-scale phenol-water extracts from each of three strains of C. matruchotii were purified by hydrophobic-interaction chromatography and shown to contain a heterogeneous lipoglycan fraction. The major fatty acids present were the same as for the whole-cell fatty acid profiles but differed in their relative amounts. Qualitative analysis of the lipoglycan fractions revealed similarities of carbohydrate composition with a previously characterized lipoglycan fraction from C. diphtheriae and with the lipoarabinomannan/lipomannans found in the genus Mycobacterium. The carbohydrate composition and the low phosphorus content indicated that lipoteichoic acid was absent from C. matruchotii. The calcium-binding properties of C. matruchotii therefore cannot be attributed to lipoteichoic acid.

Topics: Calcium; Carbohydrates; Chromatography, Thin Layer; Corynebacterium; Dental Plaque; Electrophoresis, Polyacrylamide Gel; Fatty Acids; Humans; Immunoblotting; Lipopolysaccharides; Phenols; Phosphorus; Sodium Dodecyl Sulfate; Teichoic Acids; Water