Antibiotic activity and synergistic effect of antimicrobial peptide against pathogens from a patient with gallstones

https://doi.org/10.1016/j.bbrc.2004.07.008Get rights and content

Abstract

HP (2–20) is a peptide derived from the N-terminus of Helicobacter pylori ribosomal protein L1 that has been shown to have antimicrobial activity against various species of bacteria. When we tested the effects of HP (2–20), we found that this peptide displayed strong activity against pathogens from a patient with gallstones, but it did not have hemolytic activity against human erythrocytes. We also found that HP (2–20) had potent activity against cefazolin sodium-resistant bacterial cell lines, and that HP (2–20) and cefazolin sodium had synergistic effects against cell lines resistant to the latter. To investigate the mechanism of action of HP (2–20), we performed fluorescence activated flow cytometry using pathogens from the patient with gallstones. As determined by propidium iodide (PI) staining, pathogenic bacteria treated with HP (2–20) showed higher fluorescence intensity than untreated cells, similar to melittin-treated cells, and that HP (2–20) acted in an energy- and salt-dependent manner. Scanning electron microscopy showed that HP (2–20) caused significant morphological alterations in the cell surface of pathogens from the patient with gallstones. By determining their 16S rDNA sequences, we found that both the pathogens from the patient with gallstones and the cefazolin sodium-resistant cell lines showed 100% homology with sequences from Pseudomonas aeruginosa. Taken together, these results suggest that HP (2–20) has antibiotic activity and that it may be used as a lead drug for the treatment of acquired pathogens from patients with gallstones and antibiotic-resistant cell lines.

Section snippets

Materials and methods

Peptide synthesis. The peptides were synthesized by the solid phase method using Fmoc(9-fluorenyl-methoxycarbonyl)-chemistry [22]. Rink amide 4-methyl benzhydrylamine (MBHA) resin (0.55 mmol/g) was used as the support to obtain a C-terminal amidate peptide. The coupling of Fmoc-amino acids was performed with N-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC). Amino acid side chains were protected as follows: tert-butyl (Asp), trityl (Gln), tert-butyloxycarbonyl (Lys). Deprotection

Peptide synthesis, antibacterial, and hemolytic activities

A peptide corresponding to HP (2–20)-amide was chemically synthesized, purified to homogeneity, and subjected to MALDI-mass spectroscopic analysis (Table 1). We previously showed that HP (2–20) inhibits the growth of Escherichia coli and Bacillus megaterium[20]. Melittin, a toxin from honeybee venom, has also been reported to possess potent antimicrobial activity over a broader spectrum [12]. When we compared the antibacterial MIC [25] of HP (2–20) and melittin, we found that bacteria were

Discussion

A peptide corresponding to HP (2–20) was chemically synthesized, purified to homogeneity, and subjected to MALDI-mass spectroscopy. It was found to have a molecular ion peak of 2319.3 Da, which agrees well with the calculated molecular weight (2320.0 Da). This synthetic HP (2–20) had been shown to possess potent antibacterial activity [15], [22]. When we tested it against various kinds of Gram-positive and Gram-negative bacteria, we found that HP (2–20) exhibits potent, broad-spectrum

Acknowledgments

This work was supported, in part, by grants from the Ministry of Science and Technology, Korea, and the Korea Science and Engineering Foundation through the Research Center for Proteineous Materials, and from the National R&D Program for Fusion strategy of Advanced Technologies.

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