halocidin and Hemolysis

Halocidin is an antimicrobial peptide found in the tunicate. A series of experiments were previously conducted in an attempt to develop a novel antibiotic derived from halocidin, as the peptide was determined to evidence profound antimicrobial activity against a variety of antibiotic-resistant microbes, with significantly less toxicity to human blood cells. In this study, we assessed the validity of one of the halocidin congeners, called Khal, as a new antibiotic for the treatment of systemic bacterial infections. Our in vitro antimicrobial tests showed that the MICs of Khal against several gram-positive bacteria were below 16 microg/ml in the presence of salt. We also determined that Khal retained sufficient target selectivity to discern microbial and human blood cells and was therefore capable of efficiently killing invading pathogens. Furthermore, Khal caused no aggregation problems upon incubation with human serum and also proved to be resistant to proteolysis by enzymes occurring in human serum. In the following experiments conducted with a mouse model of Listeria monocytogenes infection, we demonstrated that a single intravenous inoculation with Khal resulted in significant therapeutic effects on the survival of mice. In addition, our bacterial-enumeration analysis showed that after Listeria infection, livers and spleens from Khal-treated mice generated a great deal fewer recoverable CFU. Finally, the antibiotic effects of Khal were evaluated under confocal microscopy after we immunostained the liver sections with anti-Khal antibody. It was concluded that Khal bound specifically to the surfaces of bacteria colonized in the mouse liver and killed the bacteria rapidly.

Topics: Animals; Anti-Bacterial Agents; Blood Cells; Chromatography, High Pressure Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Hemolysis; Humans; Listeria monocytogenes; Listeriosis; Liver; Male; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Peptides; Spleen; Time Factors

Halocidin is an antimicrobial peptide, which is isolated from hemocytes from the tunicate, Halocynthiaaurantium. In this study, we cloned the full-length cDNA of halocidin from pharyngeal tissue, using a combination of RT-PCR and 5'-RACE-PCR. The observed cDNA structure indicated that halocidin is synthesized as a 10.37 kDa prepropeptide. Based on the cDNA structure and the known amino acid sequence of the mature peptide, it was concluded that the precursor of halocidin contains a 21-residue signal peptide, followed by the 18 residues of the mature peptide, and a 56-residue anionic C-terminal extension, which is removed later on in the process. The signal sequence of halocidin exhibited a high degree of similarity with the corresponding portion of the Ci-META4 protein, which had been previously discovered in the coelomic cells of another tunicate, Cionaintestinalis, and is considered to play a role in metamorphosis. However, in several respects, the cDNA structure of Ci-META4 suggested that it might constitute a precursor for an antimicrobial peptide. Thus, we prepared a synthetic peptide, which was comprised of 19 N-terminal amino acid residues in the predicted mature region of Ci-META4, and tested it with regard to its antimicrobial activity. As a result, we confirmed that the synthetic peptide exhibited potent antimicrobial activity against Gram (+) and (-) bacteria, while evidencing no hemolytic activity toward human erythrocytes.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Bacteria; Base Sequence; Cloning, Molecular; Dose-Response Relationship, Drug; Erythrocytes; Hemolysis; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Urochordata

Halocidin is a heterodimer antimicrobial peptide previously isolated from the tunicate Halocynthia aurantium. Based on the larger monomer (18Hc) of halocidin, nine halocidin congeners, including a series of 6 peptides truncated successively from the carboxyl-terminal end of 18Hc and 3 analogs (18HcKK, K19Hc, and K19HcKK), which have lysine residues in place of two internal histidines or have a lysine added to the amino terminus of the 18Hc molecule, were prepared. Each peptide was also converted into a homodimeric version. The antimicrobial activities of halocidin congeners truncated from the C terminus were dramatically decreased, suggesting that the full length of 18Hc is required for maintaining its maximum antimicrobial activity. Dimer forms of halocidin congeners exhibited stronger antimicrobial activities than the monomer of the corresponding peptide. Four dimer peptides (di-18Hc, di-18HcKK, di-K19Hc, and di-K19HcKK) were analyzed for antimicrobial activities against 10 clinically isolated antibiotic-resistant bacteria in elevated concentrations of NaCl or MgCl(2). Of the peptides studied here, di-K19Hc retained invariably strong activity against all bacteria in diverse conditions and also showed much reduced hemolytic activity against human erythrocytes.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Drug Resistance, Bacterial; Erythrocytes; Hemolysis; Humans; In Vitro Techniques; Melitten; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Urochordata