Structural and Gene Characterization of a New Antifungal Peptide Obtained from Penicillium crustosum FP11 Strain

A novel antifungal peptide, PcAFP (6.48 kDa, pI 8.83 ), was obtained from the culture supernatant of the fungus Penicillium crustosum . The gene encoding the PcAFP peptide was isolated b ased on its homologue in Penicillium chrysogenum , PgAFP. PcAFP is a small, cystine-rich peptide, and th e mature peptide consists of 58 amino acid residues. The i mmature P. crustosum antifungal protein (AFP) showed 95.65% identity to the antifungal prote in of P. chrysogenum , while the mature peptide showed 98.28% identity with PgAFP. Molecular modeling of the tertiary structure of the mature peptide revealed details of the conserved stru cture of the AFPs, such as the ? -barrel motif stabilized by three disulfide bonds and the ? -core motif. Analysis of the extract by 16% tricine SD S- PAGE showed a 6.9 kDa peptide, which was close to the pr edicted molecular mass of the mature peptide of 6.48 kDa. Assays of antimicrobial activity , performed by broth microdilution using the crude extract obtained from the culture medium, showe d activity against Candida albicans . These results demonstrate the conservation of the PcAPF gene and the high level of identity with the PgAFP antifungal protein of P. chrysogenum . Given these structural and biochemical characteristics, PcAFP could be a potential candidate for future investigations that may aid in the development of new antifungal compounds.

Isolation and characterization of an antifungal peptide from fruiting bodies of edible mushroom Lentinus squarrosulus Mont

Aims: To isolate and characterize an antimicrobial peptide from fruiting bodies of Lentinus squarrosulus Mont., the Thai common edible mushroom. Methodology and results: Solid ammonium sulfate at 40-80% (w/v) final concentration was utilized to precipitate the proteins and further purified by ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-25. The peptide was adsorbed on DEAE-cellulose and Sephadex G-25. It appeared as a single band with a molecular mass of about 17 kDa (kilodalton) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further investigation of antimicrobial properties of purified peptide revealed that it has no activity against both Gram positive and Gram negative bacteria. However, it exhibited strong antifungal activity against various species of fungal pathogen of human. Among the high sensitive strains, Trichophyton mentagrophytes, T. rubrum, and Candida tropicalis are clinical isolates. Moreover, the potency was found to be concentration dependent and comparable with Ketoconazole, the commercial antifungal drug. Conclusion, significance and impact study: In this work, the novel bioactive peptide from fruiting bodies of L. squarrosulus Mont. has been isolated. It shows potent activity against various clinical isolates of fungal pathogen of human. It may have potential for pharmaceutical application.

Cloning and purification of the first termicin-like peptide from the cockroach Eupolyphaga sinensis

Termicin is an antimicrobial peptide with six cysteines forming three disulfide bridges that was firstly isolated from the salivary glands and hemocytes of the termite Pseudacanthotermes spiniger. In contrast to many broad-spectrum antimicrobial peptides, termicin is most active against filamentous fungi. Although more than one hundred complementary DNAs (cDNAs) encoding termicin-like peptides have been reported to date, all these termicin-like peptides were obtained from Isoptera insects. Methods The cDNA was cloned by combination of cDNA library construction kit and DNA sequencing. The polypeptide was purified by gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined by Edman degradation and mass spectrometry. Antimicrobial activity was tested against several bacterial and fungal strains. The minimum inhibitory concentration (MIC) was determined by microdilution tests. Results A novel termicin-like peptide with primary structure ACDFQQCWVTCQRQYSINFISARCNGDSCVCTFRT was purified from extracts of the cockroach Eupolyphaga sinensis (Insecta: Blattodea). The cDNA encoding Es-termicin was cloned by cDNA library screening. This cDNA encoded a 60 amino acid precursor which included a 25 amino acid signal peptide. Amino acid sequence deduced from the cDNA matched well with the result of protein Edman degradation. Susceptibility test indicated that Es-termicin showed strong ability to kill fungi with a MIC of 25 g/mL against Candida albicans ATCC 90028. It only showed limited potency to affect the growth of Gram-positive bacteria with a MIC of 200 g/mL against Enterococcus faecalis ATCC 29212. It was inactive against gram-negative bacteria at the highest concentration tested (400 g/mL). Es-termicin showed high sequence similarity with termicins from many species of termites (Insecta: Isoptera). Conclusions This is the first report of a termicin-like peptide isolated from E. sinensis that belongs to the insect order Blattodea. Our results demonstrate the diversity of termicin-like peptides, as well as antimicrobial peptides in insects.

Cloning and purification of the first termicin-like peptide from the cockroach Eupolyphaga sinensis

Abstract Background Termicin is an antimicrobial peptide with six cysteines forming three disulfide bridges that was firstly isolated from the salivary glands and hemocytes of the termite Pseudacanthotermes spiniger. In contrast to many broad-spectrum antimicrobial peptides, termicin is most active against filamentous fungi. Although more than one hundred complementary DNAs (cDNAs) encoding termicin-like peptides have been reported to date, all these termicin-like peptides were obtained from Isoptera insects. Methods The cDNA was cloned by combination of cDNA library construction kit and DNA sequencing. The polypeptide was purified by gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined by Edman degradation and mass spectrometry. Antimicrobial activity was tested against several bacterial and fungal strains. The minimum inhibitory concentration (MIC) was determined by microdilution tests. Results A novel termicin-like peptide with primary structure ACDFQQCWVTCQRQYSINFISARCNGDSCVCTFRT was purified from extracts of the cockroach Eupolyphaga sinensis (Insecta: Blattodea). The cDNA encoding Es-termicin was cloned by cDNA library screening. This cDNA encoded a 60 amino acid precursor which included a 25 amino acid signal peptide. Amino acid sequence deduced from the cDNA matched well with the result of protein Edman degradation. Susceptibility test indicated that Es-termicin showed strong ability to kill fungi with a MIC of 25 μg/mL against Candida albicans ATCC 90028. It only showed limited potency to affect the growth of Gram-positive bacteria with a MIC of 200 μg/mL against Enterococcus faecalis ATCC 29212. It was inactive against gram-negative bacteria at the highest concentration tested (400 μg/mL). Es-termicin showed high sequence similarity with termicins from many species of termites (Insecta: Isoptera). Conclusions This is the first report of a termicin-like peptide isolated from E. sinensis that belongs to the insect order Blattodea. Our results demonstrate the diversity of termicin-like peptides, as well as antimicrobial peptides in insects.(AU)