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Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides.

Abraham P, Sundaram A, R A, V R, George S, Kumar KS - PLoS ONE (2015)

Bottom Line: Its higher activity was well correlated with the improved inner membrane permeabilisation capacity.The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane.Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5.

View Article: PubMed Central - PubMed

Affiliation: Chemical Biology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.

ABSTRACT
All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.

No MeSH data available.


Related in: MedlinePlus

Outer and inner membrane permeabilisation in E. coli cells.(A) E. coli cells(OD 0.5–0.6) were washed with 5mM HEPES, pH 7.2 and 5mM KCN. 1mL cells were treated with 10mM NPN and increasing concentrations of peptides were added. The uptake of NPN was noted by increase in fluorescence at an excitation of 350nm and emission of 420nmas a measure of outer membrane permeabilisation. (B) Inner membrane permeabilisation efficiency of the peptides were assayed by treating E. coli cells (OD 0.4–0.6) in PBS with 1μM SYTOX green at 37°C for 15 min. Fluorescence was measured at an excitation wavelength of 485nm and emission wavelength of 520nm after peptide addition.
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pone.0124210.g003: Outer and inner membrane permeabilisation in E. coli cells.(A) E. coli cells(OD 0.5–0.6) were washed with 5mM HEPES, pH 7.2 and 5mM KCN. 1mL cells were treated with 10mM NPN and increasing concentrations of peptides were added. The uptake of NPN was noted by increase in fluorescence at an excitation of 350nm and emission of 420nmas a measure of outer membrane permeabilisation. (B) Inner membrane permeabilisation efficiency of the peptides were assayed by treating E. coli cells (OD 0.4–0.6) in PBS with 1μM SYTOX green at 37°C for 15 min. Fluorescence was measured at an excitation wavelength of 485nm and emission wavelength of 520nm after peptide addition.

Mentions: 1-N-phenylnapthylamine (NPN) is a neutral hydrophobic probe that is normally excluded by an intact outer membrane, but can penetrate into the cell upon membrane disruption by membrane active peptide like polymyxin B and produces an intense fluorescence. The NPN uptake was observed when treated with SMMD-B1CTcu5 analogs except in the case of B1CTcu5-Rana and B1CTcu5P11-C21. The effects induced by these peptides was observed at concentrations well below their respective MICs (Fig 3A).


Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides.

Abraham P, Sundaram A, R A, V R, George S, Kumar KS - PLoS ONE (2015)

Outer and inner membrane permeabilisation in E. coli cells.(A) E. coli cells(OD 0.5–0.6) were washed with 5mM HEPES, pH 7.2 and 5mM KCN. 1mL cells were treated with 10mM NPN and increasing concentrations of peptides were added. The uptake of NPN was noted by increase in fluorescence at an excitation of 350nm and emission of 420nmas a measure of outer membrane permeabilisation. (B) Inner membrane permeabilisation efficiency of the peptides were assayed by treating E. coli cells (OD 0.4–0.6) in PBS with 1μM SYTOX green at 37°C for 15 min. Fluorescence was measured at an excitation wavelength of 485nm and emission wavelength of 520nm after peptide addition.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440778&req=5

pone.0124210.g003: Outer and inner membrane permeabilisation in E. coli cells.(A) E. coli cells(OD 0.5–0.6) were washed with 5mM HEPES, pH 7.2 and 5mM KCN. 1mL cells were treated with 10mM NPN and increasing concentrations of peptides were added. The uptake of NPN was noted by increase in fluorescence at an excitation of 350nm and emission of 420nmas a measure of outer membrane permeabilisation. (B) Inner membrane permeabilisation efficiency of the peptides were assayed by treating E. coli cells (OD 0.4–0.6) in PBS with 1μM SYTOX green at 37°C for 15 min. Fluorescence was measured at an excitation wavelength of 485nm and emission wavelength of 520nm after peptide addition.
Mentions: 1-N-phenylnapthylamine (NPN) is a neutral hydrophobic probe that is normally excluded by an intact outer membrane, but can penetrate into the cell upon membrane disruption by membrane active peptide like polymyxin B and produces an intense fluorescence. The NPN uptake was observed when treated with SMMD-B1CTcu5 analogs except in the case of B1CTcu5-Rana and B1CTcu5P11-C21. The effects induced by these peptides was observed at concentrations well below their respective MICs (Fig 3A).

Bottom Line: Its higher activity was well correlated with the improved inner membrane permeabilisation capacity.The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane.Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5.

View Article: PubMed Central - PubMed

Affiliation: Chemical Biology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.

ABSTRACT
All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.

No MeSH data available.


Related in: MedlinePlus