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Discovery of Novel Bacterial Cell-Penetrating Phylloseptins in Defensive Skin Secretions of the South American Hylid Frogs, Phyllomedusa duellmani and Phyllomedusa coelestis

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ABSTRACT

Phylloseptin (PS) peptides, derived from South American hylid frogs (subfamily Phyllomedusinae), have been found to have broad-spectrum antimicrobial activities and relatively low haemolytic activities. Although PS peptides have been identified from several well-known and widely-distributed species of the Phyllomedusinae, there remains merit in their study in additional, more obscure and specialised members of this taxon. Here, we report the discovery of two novel PS peptides, named PS-Du and PS-Co, which were respectively identified for the first time and isolated from the skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis. Their encoding cDNAs were cloned, from which it was possible to deduce the entire primary structures of their biosynthetic precursors. Reversed-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) analyses were employed to isolate and structurally-characterise respective encoded PS peptides from skin secretions. The peptides had molecular masses of 2049.7 Da (PS-Du) and 1972.8 Da (PS-Co). They shared typical N-terminal sequences and C-terminal amidation with other known phylloseptins. The two peptides exhibited growth inhibitory activity against E. coli (NCTC 10418), as a standard Gram-negative bacterium, S. aureus (NCTC 10788), as a standard Gram-positive bacterium and C. albicans (NCPF 1467), as a standard pathogenic yeast, all as planktonic cultures. Moreover, both peptides demonstrated the capability of eliminating S. aureus biofilm.

No MeSH data available.


Reverse phase HPLC chromatogram of the skin secretion of Phyllomedusa coelestis (A) with arrow showing the absorbance peak corresponding to natural PS-Co. Reverse phase HPLC profile of synthetic PS-Co (B) with absorbance peak at relevant position to that in (A); Co-elution reverse phase HPLC chromatogram of synthetic PS-Co added to crude skin secretion of Phyllomedusa coelestis (C) with absorbance peak at relevant position to that in (A). The Y-axis shows the relative absorbance in absorbance units at 214 nm and the X-axis shows the retention time in minutes.
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toxins-08-00255-f005: Reverse phase HPLC chromatogram of the skin secretion of Phyllomedusa coelestis (A) with arrow showing the absorbance peak corresponding to natural PS-Co. Reverse phase HPLC profile of synthetic PS-Co (B) with absorbance peak at relevant position to that in (A); Co-elution reverse phase HPLC chromatogram of synthetic PS-Co added to crude skin secretion of Phyllomedusa coelestis (C) with absorbance peak at relevant position to that in (A). The Y-axis shows the relative absorbance in absorbance units at 214 nm and the X-axis shows the retention time in minutes.

Mentions: The lyophilized crude skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis were respectively fractioned by reversed-phase high-performance liquid chromatography (RP-HPLC) and the chromatograms are shown in Figure 4A and Figure 5A, with arrows indicating the retention times/elution positions of peptides with masses coincident with the approximate predicted molecular masses of PS-Du and PS-Co. The HPLC elution profile of synthetic PS-Du and its co-elution profile with the crude skin secretion of Phyllomedusa duellmani is shown in Figure 4B,C. Likewise, the HPLC elution profile of synthetic PS-Co and its co-elution profile with the crude skin secretion of Phyllomedusa coelestis is shown in Figure 5B,C. The masses of the peptides in fractions corresponding to PS-Du and PS-Co were detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a linear time-of-flight Voyager DE mass spectrometer (Perceptive Biosystem, Bedford, MA, USA) (Figure 6). The amino acid sequence of PS-Du and PS-Co were further analysed by MS/MS fragmentation sequencing shown in Figure 7A, B. The amino acid sequences of the mature peptides, PS-Du and PS-Co, were thus unequivocally identified and the glycine (G) residue at the carboxyl terminus of both precursors was also confirmed as an amide donor.


Discovery of Novel Bacterial Cell-Penetrating Phylloseptins in Defensive Skin Secretions of the South American Hylid Frogs, Phyllomedusa duellmani and Phyllomedusa coelestis
Reverse phase HPLC chromatogram of the skin secretion of Phyllomedusa coelestis (A) with arrow showing the absorbance peak corresponding to natural PS-Co. Reverse phase HPLC profile of synthetic PS-Co (B) with absorbance peak at relevant position to that in (A); Co-elution reverse phase HPLC chromatogram of synthetic PS-Co added to crude skin secretion of Phyllomedusa coelestis (C) with absorbance peak at relevant position to that in (A). The Y-axis shows the relative absorbance in absorbance units at 214 nm and the X-axis shows the retention time in minutes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037481&req=5

toxins-08-00255-f005: Reverse phase HPLC chromatogram of the skin secretion of Phyllomedusa coelestis (A) with arrow showing the absorbance peak corresponding to natural PS-Co. Reverse phase HPLC profile of synthetic PS-Co (B) with absorbance peak at relevant position to that in (A); Co-elution reverse phase HPLC chromatogram of synthetic PS-Co added to crude skin secretion of Phyllomedusa coelestis (C) with absorbance peak at relevant position to that in (A). The Y-axis shows the relative absorbance in absorbance units at 214 nm and the X-axis shows the retention time in minutes.
Mentions: The lyophilized crude skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis were respectively fractioned by reversed-phase high-performance liquid chromatography (RP-HPLC) and the chromatograms are shown in Figure 4A and Figure 5A, with arrows indicating the retention times/elution positions of peptides with masses coincident with the approximate predicted molecular masses of PS-Du and PS-Co. The HPLC elution profile of synthetic PS-Du and its co-elution profile with the crude skin secretion of Phyllomedusa duellmani is shown in Figure 4B,C. Likewise, the HPLC elution profile of synthetic PS-Co and its co-elution profile with the crude skin secretion of Phyllomedusa coelestis is shown in Figure 5B,C. The masses of the peptides in fractions corresponding to PS-Du and PS-Co were detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a linear time-of-flight Voyager DE mass spectrometer (Perceptive Biosystem, Bedford, MA, USA) (Figure 6). The amino acid sequence of PS-Du and PS-Co were further analysed by MS/MS fragmentation sequencing shown in Figure 7A, B. The amino acid sequences of the mature peptides, PS-Du and PS-Co, were thus unequivocally identified and the glycine (G) residue at the carboxyl terminus of both precursors was also confirmed as an amide donor.

View Article: PubMed Central - PubMed

ABSTRACT

Phylloseptin (PS) peptides, derived from South American hylid frogs (subfamily Phyllomedusinae), have been found to have broad-spectrum antimicrobial activities and relatively low haemolytic activities. Although PS peptides have been identified from several well-known and widely-distributed species of the Phyllomedusinae, there remains merit in their study in additional, more obscure and specialised members of this taxon. Here, we report the discovery of two novel PS peptides, named PS-Du and PS-Co, which were respectively identified for the first time and isolated from the skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis. Their encoding cDNAs were cloned, from which it was possible to deduce the entire primary structures of their biosynthetic precursors. Reversed-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) analyses were employed to isolate and structurally-characterise respective encoded PS peptides from skin secretions. The peptides had molecular masses of 2049.7 Da (PS-Du) and 1972.8 Da (PS-Co). They shared typical N-terminal sequences and C-terminal amidation with other known phylloseptins. The two peptides exhibited growth inhibitory activity against E. coli (NCTC 10418), as a standard Gram-negative bacterium, S. aureus (NCTC 10788), as a standard Gram-positive bacterium and C. albicans (NCPF 1467), as a standard pathogenic yeast, all as planktonic cultures. Moreover, both peptides demonstrated the capability of eliminating S. aureus biofilm.

No MeSH data available.