lipoteichoic-acid and Hemolysis

Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system, offering protection against invading pathogenic bacteria. In nature, AMPs serve as antibiotics with broad-spectrum antimicrobial and anti-biofilm properties. However, low effective stability in high-salt environments and physiological instability in biological membranes limit the applicability of naturally occurring AMPs as novel therapeutics. We therefore designed short synthetic cationic peptides by substituting key residues in myxinidin, an AMP derived from the epidermal mucus of hagfish, with lysine (Lys, K), arginine (Arg, R), and tryptophan (Trp, W). The resultant myxinidin analogs exhibited strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains, even under high-salt conditions. Moreover, these peptides showed high binding affinity for both lipopolysaccharides and lipoteichoic acids and inhibited biofilm formation by most bacteria, but did not cause significant lysis of human red blood cells and were not cytotoxic to normal human keratinocytes. Circular dichroism analysis revealed that myxinidin and its analogs assumed α-helical or β-sheet structures within artificial liposomes and bacterial membranes. In addition, bacterial killing and membrane permeation experiments demonstrated that the myxinidin analogs permeated through bacterial membranes, leading to cytoplasmic disruption and cell death. Taken together, these findings suggest myxinidin analogs may be promising candidate antibiotic agents for therapeutic application against antibiotic-resistant bacteria.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Biofilms; Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Circular Dichroism; Drug Design; Erythrocytes; Fish Proteins; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Humans; Keratinocytes; Lipopolysaccharides; Microbial Sensitivity Tests; Oligopeptides; Protein Binding; Salt Tolerance; Structure-Activity Relationship; Teichoic Acids

The emergence of multi-drug resistant (MDR) microbes leads to urgent demands for novel antibiotics exploration. We demonstrated a cDNA from amphioxus Branchiostoma japonicum, designated Bjamp1, encoded a protein with features typical of antimicrobial peptides (AMPs), which is not homologous to any AMPs currently discovered. It was found that Bjamp1 was expressed in distinct tissues, and its expression was remarkably up-regulated following challenge with LPS and LTA. Moreover, the synthesized putative mature AMP, mBjAMP1, underwent a coil-to-helix transition in the presence of TFE or SDS, agreeing well with the expectation that BjAMP1 was a potential AMP. Functional assays showed that mBjAMP1 inhibited the growth of all the bacteria tested, and induced membrane/cytoplasmic damage. ELISA indicated that mBjAMP1 was a pattern recognition molecule capable of identifying LPS and LTA. Importantly, mBjAMP1 disrupted the bacterial membranes by a membranolytic mechanism. Additionally, mBjAMP1 was non-cytotoxic to mammalian cells. Collectively, these data indicate that mBjAMP1 is a new AMP with a high bacterial membrane selectivity, rendering it a promising template for the design of novel peptide antibiotics against MDR microbes. It also shows for the first time that use of signal conserved sequence of AMPs is effective identifying potential AMPs across different animal classes.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacteria; Cell Line; Cell Survival; Circular Dichroism; Databases, Genetic; Erythrocytes; Hemolysis; Humans; Lancelets; Lipopolysaccharides; Mice; Microbial Sensitivity Tests; Microscopy, Electron, Transmission; Molecular Sequence Data; Protein Binding; Teichoic Acids

Interactions of bacterial and host products in activating the innate immune system is an important area to address. The role of lipoteichoic acid (LTA) in these interactions is particularly important because it is understudied in comparison to other factors. This study evaluated the effect of cationic peptides (CPs) on LTA-induced proinflammatory cytokine production in human whole blood and on purified leukocytes. Four different CPs of truncated derivatives from the known peptides LL37, BPI, and CP207 were used. Two of the CPs (IG33 and LL33), derivatives from LL37, potentiated S. aureus LTA induced TNFα, IL-6 and IL-1β production in whole blood. The release of TNFα was increased 30-fold after 16 hours incubation. Intact LL37 also increased LTA-induced TNFα and IL-1β in a time dependent manner. LTA in combination with either LL33 or IG23 demonstrated a synergistic enhanced TNFα and IL-1β secretion on isolated leukocytes but not on purified monocytes. When complexed with IG23 and LL33, the electrophoretic mobility of LTA was altered in a non-denaturating gel electrophoresis. LTA was disaggregated and migrated more rapidly, suggesting an amphiphilic effect of CPs on LTA. In conclusion, LTA synergizes with LL37 and its truncated derivatives and this may lead to proinflammatory cytokine production and cause problems in sepsis therapy.

Topics: Antimicrobial Cationic Peptides; Cathelicidins; Cell Survival; Cytokines; Electrophoresis, Polyacrylamide Gel; Hemoglobins; Hemolysis; Humans; Inflammation Mediators; Leukocytes; Lipopolysaccharides; Monocytes; Mutant Proteins; Teichoic Acids

The goal of this study was to evaluate the effects of DNA and F-actin [polyanions present in high concentration in cystic fibrosis (CF) airway fluid] on the antibacterial activities of the cationic steroid antibiotic CSA-13 and the cationic peptides LL37, WLBU2 and HB71.. Light scattering intensity was used to evaluate the aggregation of DNA and F-actin by the cationic antibacterial agents. Bacterial killing assays, atomic force microscopy, determination of MIC values and bacterial load of CF sputa were used to determine the bactericidal activity. Inhibition of nuclear factor-kappaB (NF-kappaB) translocation in human aorta endothelial cells (HAECs) was quantified as an assay of anti-inflammatory action.. CSA-13 is significantly more effective than cationic antibacterial peptides against kanamycin-resistant Pseudomonas aeruginosa and less susceptible to inactivation by DNA or F-actin. The concentration of CSA-13 sufficient to decrease the CF sputa bacteria load by approximately 90% is at least 10 times lower than that at which CSA-13 formed aggregates with DNA or F-actin. Both CSA-13 and LL37 prevent lipopolysaccharide-induced translocation of NF-kappaB in HAEC, thereby suggesting that these antibacterial molecules might prevent systemic inflammation caused by bacterial wall components.. Charge-based interactions that strongly inhibit the antibacterial activity of host cationic antibacterial peptides present in CF sputa have significantly less effect on molecules from the ceragenin family such as CSA-13 due in part to their smaller net charge and distribution of this charge over a hydrophobic scaffold. CSA molecules therefore have potential for the treatment of chronic infections and inflammation that occur in CF airways and other settings in which extracellular polyanions accumulate.

Topics: Actins; Adult; Amino Acid Sequence; Anti-Bacterial Agents; Bacteria; Cell Line; Cystic Fibrosis; Deoxyribonuclease I; DNA; Drug Resistance, Bacterial; Electrolytes; Endothelial Cells; Erythrocytes; Hemolysis; Humans; Lipopolysaccharides; Microbial Sensitivity Tests; Microscopy, Atomic Force; Peptides; Sputum; Steroids; Teichoic Acids

We studied three model antibacterial peptides that resembled the N-terminal 18 amino acids of SMAP-29, an alpha-helical, antimicrobial peptide of sheep. Although the parent compound, ovispirin-1 (KNLRR IIRKI IHIIK KYG), was potently antimicrobial, it was also highly cytotoxic to human epithelial cells and hemolytic for human erythrocytes. Single residue substitutions to ovispirin-1 yielded two substantially less cytotoxic peptides (novispirins), with intact antimicrobial properties. One of these, novispirin G-10, differed from ovispirin-1 only by containing glycine at position 10, instead of isoleucine. The other, novispirin T-7, contained threonine instead of isoleucine at position 7. We determined the three-dimensional solution structures of all three peptides by circular dichroism spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy. Although all retained an amphipathic helical structure in 2,2,2-trifluoroethanol, they manifested subtle fine-structural changes that evidently impacted their activities greatly. These findings show that simple structural modifications can 'fine-tune' an antimicrobial peptide to minimize unwanted cytotoxicity while retaining its desired activity.

Topics: Amino Acid Substitution; Antimicrobial Cationic Peptides; Cell Survival; Drug Design; Erythrocytes; Hemolysis; Humans; Lipopolysaccharides; Mutation; Protein Conformation; Solutions; Structure-Activity Relationship; Teichoic Acids; Trifluoroethanol

Human neutrophils which are pretreated with subtoxic concentrations of a variety of lysophosphatides (lysophosphatidylcholine, lysophosphatidylcholine oleoyl, lysophosphatidylcholine myrioyl, lysophosphatidylcholine stearoyl, lysophosphatidylcholine gamma-O-hexadecyl, lysophosphatidylinositol, and lysophosphatidylglycerol) act synergistically with neutrophil agonists phorbol myristate acetate, immune complexes, poly-L-histidine, phytohemagglutinin, and N-formyl-methionyl-leucyl-phenyalanine to cause enhanced generation of superoxide (O2-). None of the lyso compounds by themselves caused generation of O2-. The lyso compounds strongly bound to the neutrophils and could not be washed away. All of the lyso compounds that collaborated with agonists to stimulate O2- generation were hemolytic for human red blood cells. On the other hand, lyso compounds that were nonhemolytic for red blood cells (lysophosphatidylcholine caproate, lysophosphatidylcholine decanoyl, lysophosphatidylethanolamine, lysophosphatidylserine) failed to collaborate with agonists to generate synergistic amounts of O2-. However, in the presence of cytochalasin B, both lysophosphatidylethanolamine and lysophosphatidylserine also markedly enhanced O2- generation induced by immune complexes. O2- generation was also very markedly enhanced when substimulatory amounts of arachidonic acid or eicosapentanoic acid were added to PMNs in the presence of a variety of agonists. On the other hand, neither phospholipase C, streptolysin S (highly hemolytic), phospholipase A2, phosphatidylcholine, nor phosphatidylcholine dipalmitoyl (all nonhemolytic) had the capacity to synergize with any of the agonists tested to generate enhanced amounts of O2-. The data suggest that in addition to long-chain fatty acids, only those lyso compounds that possess fatty acids with more than 10 carbons and that are also highly hemolytic can cause enhanced generation of O2- in stimulated PMNs.

Topics: Adjuvants, Immunologic; Antigen-Antibody Complex; Hemolysis; Histidine; Humans; In Vitro Techniques; Lipopolysaccharides; Lysophospholipids; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peptides; Phytohemagglutinins; Superoxides; Teichoic Acids; Tetradecanoylphorbol Acetate

Lipoteichoic acids (LTA) released by gram-positive bacteria can spontaneously bind to mammalian cell surfaces. In the present study, erythrocytes (E) sensitized with pneumococcal LTA (LTA-E) were used as a model system to determine if LTA could render host cells susceptible to damage by autologous complement. Complement (C)-mediated lysis of LTA-E from normal rats and normal humans occurred when these cells were incubated in their respective autologous sera in vitro. In addition, when LTA-E from a C2-deficient human and from C4-deficient guinea pigs were incubated in their autologous sera, there was significant lysis in vitro, demonstrating a role for the alternative pathway. The in vivo survival of 51Cr-labeled autologous LTA-E was also studied. Only 2.9% of autologous LTA-E remained in the circulation of normal rats after 90 min. In contrast, 31.2% of autologous LTA-E remained in the circulation of rats depleted of C3. Intravascular hemolysis accounted for the clearance of LTA-E in the normal rats, whereas liver sequestration was responsible for clearance in the C3-depleted rats. These results demonstrate that LTA can render the host's cells susceptible to damage by its own complement system, establishing this as a possible mechanism of tissue damage in natural bacterial infections.

Topics: Animals; Bacterial Infections; Complement Activation; Complement System Proteins; Egtazic Acid; Elapid Venoms; Guinea Pigs; Hemolysis; Humans; Lipopolysaccharides; Male; Phosphatidic Acids; Rats; Rats, Inbred Strains; Teichoic Acids

Previous studies have demonstrated that lipoteichoic acid (LTA) from Streptococcus pneumoniae binds to erythrocytes and renders them susceptible to lysis by autologous complement. The present study was performed to determine whether LTA from two other gram-positive bacterial species had the ability to render mammalian cells susceptible to lysis by autologous complement. Human erythrocytes were sensitized with LTA from S. pneumoniae, Streptococcus pyogenes, or Lactobacillus fermentum. Under incubation in normal autologous serum, lysis was observed with each of the LTA-sensitized erythrocyte preparations. When erythrocytes from a C2-deficient patient were sensitized with the LTA preparations and then incubated in autologous, C2-deficient serum, the erythrocytes sensitized with S. pyogenes or L. fermentum LTA demonstrated relatively little lysis, whereas the erythrocytes sensitized with S. pneumoniae LTA yielded near-total lysis. After reconstitution of the C2-deficient serum with purified human C2, lysis was observed with all three LTA preparations. When erythrocytes from an agammaglobulinemic patient were sensitized with either the S. pyogenes or the L. fermentum LTA, they were not lysed in the presence of autologous agammaglobulinemic serum, whereas the erythrocytes sensitized with S. pneumoniae LTA were completely lysed. Serum obtained from the agammaglobulinemic patient after reconstitution with intravenous pooled gamma globulin was able to lyse autologous erythrocytes sensitized with each of the three LTA preparations. These results demonstrate that the ability to render host cells susceptible to lysis by autologous complement is a general property of LTA. Whether activation of the autologous complement occurs by the classical or alternative pathways and whether it is antibody dependent depends on the nature of the bacterial LTA.

Topics: Agammaglobulinemia; Complement C2; Erythrocytes; Hemolysis; Humans; Immunoglobulin G; Lactobacillus; Lipopolysaccharides; Phosphatidic Acids; Streptococcus pneumoniae; Streptococcus pyogenes; Teichoic Acids

The critical micelle concentration (CMC) of lipoteichoic acid (LTA) was investigated with two dyes, rhodamine 6G and Coomassie brilliant blue R-250. Both dyes gave similar values for the CMC of LTA. The CMC of LTA from several species of bacteria ranged from 28 to 60 micrograms/ml in phosphate-buffered saline. The CMC values for the LTAs are in the range expected for an amphiphile containing a single, 16-carbon fatty acid residue. Formation of micelles was not detected with deacylated LTA. Salt decreased the CMC of LTA from 380 micrograms/ml in distilled water to 37 micrograms/ml in 0.5 M NaCl. At concentrations greater than the CMC, LTA induced the lysis of sheep erythrocytes and was cytotoxic for Girardi heart cells. The data suggest that LTA in the micellar state may cause disruption of the erythrocyte membrane and may be cytotoxic for cells in culture.

Topics: Animals; Colloids; Hemolysis; Humans; Lipopolysaccharides; Micelles; Phosphatidic Acids; Rhodamines; Rosaniline Dyes; Sheep; Teichoic Acids