tachyplesin-peptide--tachypleus-tridentatus and Neutropenia

We investigated the efficacy of tazobactam/piperacillin (TZP), tachyplesin III and granulocyte-colony stimulating factor (G-CSF) in an experimental murine neutropenic intraabdominal infection. BALB/c male mice were rendered neutropenic by intraperitoneal administration of cyclophosphamide on days -4 and -2 pre-infection. Septic shock was induced by cecal ligation and puncture. Animals received intravenously isotonic sodium chloride solution (control group C1), 1mg/kg of tachyplesin III, 120 mg/kg of TZP, 0.1mg/kg of G-CSF, tachyplesin III plus TZP, G-CSF plus TZP and finally tachyplesin III plus G-CSF plus TZP, respectively. Lethality, bacterial growth in blood, peritoneum, spleen, liver, and mesenteric lymph nodes, endotoxin, IL-6 and TNF-alpha concentrations in plasma were evaluated. All compounds reduced the lethality when compared to controls. Endotoxin and cytokine plasma levels were significantly higher in TZP-treated animals compared to tachyplesin III-treated animals. Finally, all drug combinations showed to be the most effective treatment in reducing all variables measured. Interestingly, the strongest results concerning the bacterial growth inhibition, lethality and endotoxemia were obtained when the three compounds were contemporaneously administered. The presence of their positive interaction makes tachyplesin III and G-CSF potentially valuable as an adjuvant for antimicrobial chemotherapy of sepsis.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Disease Models, Animal; DNA-Binding Proteins; Drug Synergism; Enterococcus faecalis; Escherichia coli; Granulocyte Colony-Stimulating Factor; Male; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Neutropenia; Penicillanic Acid; Peptides, Cyclic; Peritonitis; Piperacillin; Survival Rate; Tazobactam; Time Factors

Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationic antimicrobial peptide polyphemusin I were designed with improved amphipathic profiles. Circular dichroism spectroscopy analysis indicated that in phosphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a type II beta-turn-rich structure, but, like polyphemusin I, all three variants adopted a typical beta-sheet structure in an anionic lipid environment. Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations. The variants were moderately less active in vitro but more effective in animal models. Moreover, these variants exhibited delayed bacterial killing, whereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 microg/mL. All the peptides showed similar abilities to bind to bacterial lipopolysaccharide (LPS) and permeabilize bacterial outer membranes. Consistent with this was the observation that all peptides significantly inhibited cytokine production by LPS-stimulated macrophages and penetrated polyanionic LPS monolayers to similar extents. None of the peptides had affinity for neutral lipids as evident from both tryptophan fluorescence spectroscopy and Langmuir monolayer analysis. As compared to polyphemusin I, all variants showed reduced ability to interact with anionic lipids, and the hemolytic activity of the variants was decreased by 2-4-fold. In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E. coli, as assessed using a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocyanine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability. The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potential by polyphemusin I occurred prior to lethal damage to cells. Our data suggest that increase of amphipathicity of beta-sheet polyphemusin I generally resulted in variants with decreased activity for membranes. Interestingly, all variants showed an improved ability to protect mice both against infection by Pseudomonas aeruginosa and from endotoxaemia.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacterial Outer Membrane Proteins; Cell Membrane Permeability; Circular Dichroism; Disease Models, Animal; DNA-Binding Proteins; Endotoxins; Female; Hemolysin Proteins; Horseshoe Crabs; Humans; Injections, Intraperitoneal; Lipopolysaccharides; Liposomes; Mice; Microbial Sensitivity Tests; Neutropenia; Peptides, Cyclic; Phospholipids; Protein Binding; Protein Engineering; Protein Isoforms; Protein Structure, Secondary; Sequence Homology, Amino Acid; Structure-Activity Relationship