Limits...
Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa.

Bellemare A, Vernoux N, Morin S, Gagné SM, Bourbonnais Y - BMC Microbiol. (2010)

Bottom Line: The N-terminal cementoin domain adopts α-helical secondary structures in a membrane mimetic environment, which is common in antimicrobial peptides.However, unlike numerous linear polycationic antimicrobial peptides, membrane disruption does not appear to be the main function of either cementoin, elafin or full-length pre-elafin/trappin-2 against P. aeruginosa.Our results rather suggest that pre-elafin/trappin-2 and elafin, but not cementoin, possess the ability to modulate the expression of some P.aeruginosa virulence factors, possibly through acting on intracellular targets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Biochimie, Microbiologie et Bio-informatique, Institut de Biologie Intégrative et des Systèmes and Regroupement PROTEO, Université Laval, Québec, Canada.

ABSTRACT

Background: Pre-elafin/trappin-2 is a human innate defense molecule initially described as a potent inhibitor of neutrophil elastase. The full-length protein as well as the N-terminal "cementoin" and C-terminal "elafin" domains were also shown to possess broad antimicrobial activity, namely against the opportunistic pathogen P. aeruginosa. The mode of action of these peptides has, however, yet to be fully elucidated. Both domains of pre-elafin/trappin-2 are polycationic, but only the structure of the elafin domain is currently known. The aim of the present study was to determine the secondary structures of the cementoin domain and to characterize the antibacterial properties of these peptides against P. aeruginosa.

Results: We show here that the cementoin domain adopts an α-helical conformation both by circular dichroism and nuclear magnetic resonance analyses in the presence of membrane mimetics, a characteristic shared with a large number of linear polycationic antimicrobial peptides. However, pre-elafin/trappin-2 and its domains display only weak lytic properties, as assessed by scanning electron micrography, outer and inner membrane depolarization studies with P. aeruginosa and leakage of liposome-entrapped calcein. Confocal microscopy of fluorescein-labeled pre-elafin/trappin-2 suggests that this protein possesses the ability to translocate across membranes. This correlates with the finding that pre-elafin/trappin-2 and elafin bind to DNA in vitro and attenuate the expression of some P. aeruginosa virulence factors, namely the biofilm formation and the secretion of pyoverdine.

Conclusions: The N-terminal cementoin domain adopts α-helical secondary structures in a membrane mimetic environment, which is common in antimicrobial peptides. However, unlike numerous linear polycationic antimicrobial peptides, membrane disruption does not appear to be the main function of either cementoin, elafin or full-length pre-elafin/trappin-2 against P. aeruginosa. Our results rather suggest that pre-elafin/trappin-2 and elafin, but not cementoin, possess the ability to modulate the expression of some P.aeruginosa virulence factors, possibly through acting on intracellular targets.

Show MeSH

Related in: MedlinePlus

Biophysical characterization of cementoin. A) CD spectra of cementoin with varying concentrations of TFE (up to 75%). The vertical lines indicate 208 and 222 nm, i.e. characteristic wavelengths for assessing the presence of α-helices. B) 2 D 15N-HSQC spectrum of cementoin in the presence of 50% TFE. Backbone assignments are shown. Side-chain Asn, Gln and Arg doublets are depicted with a line between the two resonances while unassigned additional peaks (potentially arising from slow exchange, see text) are labeled by an asterisk (*). C) SSP analysis of backbone Cα and Cβ chemical shifts. Secondary structure predictions depend on the result where positive values infer α-helices and negative values, β-sheets. D) Secondary structure predictions from AGADIR with α-helices shown as black boxes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2958999&req=5

Figure 1: Biophysical characterization of cementoin. A) CD spectra of cementoin with varying concentrations of TFE (up to 75%). The vertical lines indicate 208 and 222 nm, i.e. characteristic wavelengths for assessing the presence of α-helices. B) 2 D 15N-HSQC spectrum of cementoin in the presence of 50% TFE. Backbone assignments are shown. Side-chain Asn, Gln and Arg doublets are depicted with a line between the two resonances while unassigned additional peaks (potentially arising from slow exchange, see text) are labeled by an asterisk (*). C) SSP analysis of backbone Cα and Cβ chemical shifts. Secondary structure predictions depend on the result where positive values infer α-helices and negative values, β-sheets. D) Secondary structure predictions from AGADIR with α-helices shown as black boxes.

Mentions: Different experiments were performed to characterize the structure of cementoin and its interaction with membranes. First, we recorded circular dichroism (CD) spectra in the presence or absence of trifluoroethanol (TFE), which mimics a membrane environment [33] (Fig. 1A). In an aqueous solution, the CD spectrum is typical of an unstructured protein with a prominent negative peak at 199 nm. When TFE was added, the intensity of this peak decreased concomitantly with the appearance of minima around 205 nm and 222 nm whose intensity increased with the concentration of TFE. This is characteristic of an α-helical structure and the α-helical content of cementoin was estimated to be 48% in 50% TFE and up to 58% in 75% TFE. The observed isodichroic point at 203 nm indicates that the transition between the unstructured to the α-helical conformation is a two-state transition. Hence, a hydrophobic environment either induces or stabilizes α-helices in cementoin. This is in agreement with the AGADIR algorithm (Fig. 1D), which predicts the formation of two α-helices in cementoin: helix 1 with residues 10- 16 and helix 2 with residues 24-31, for a predicted total α-helical content of 39%.


Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa.

Bellemare A, Vernoux N, Morin S, Gagné SM, Bourbonnais Y - BMC Microbiol. (2010)

Biophysical characterization of cementoin. A) CD spectra of cementoin with varying concentrations of TFE (up to 75%). The vertical lines indicate 208 and 222 nm, i.e. characteristic wavelengths for assessing the presence of α-helices. B) 2 D 15N-HSQC spectrum of cementoin in the presence of 50% TFE. Backbone assignments are shown. Side-chain Asn, Gln and Arg doublets are depicted with a line between the two resonances while unassigned additional peaks (potentially arising from slow exchange, see text) are labeled by an asterisk (*). C) SSP analysis of backbone Cα and Cβ chemical shifts. Secondary structure predictions depend on the result where positive values infer α-helices and negative values, β-sheets. D) Secondary structure predictions from AGADIR with α-helices shown as black boxes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2958999&req=5

Figure 1: Biophysical characterization of cementoin. A) CD spectra of cementoin with varying concentrations of TFE (up to 75%). The vertical lines indicate 208 and 222 nm, i.e. characteristic wavelengths for assessing the presence of α-helices. B) 2 D 15N-HSQC spectrum of cementoin in the presence of 50% TFE. Backbone assignments are shown. Side-chain Asn, Gln and Arg doublets are depicted with a line between the two resonances while unassigned additional peaks (potentially arising from slow exchange, see text) are labeled by an asterisk (*). C) SSP analysis of backbone Cα and Cβ chemical shifts. Secondary structure predictions depend on the result where positive values infer α-helices and negative values, β-sheets. D) Secondary structure predictions from AGADIR with α-helices shown as black boxes.
Mentions: Different experiments were performed to characterize the structure of cementoin and its interaction with membranes. First, we recorded circular dichroism (CD) spectra in the presence or absence of trifluoroethanol (TFE), which mimics a membrane environment [33] (Fig. 1A). In an aqueous solution, the CD spectrum is typical of an unstructured protein with a prominent negative peak at 199 nm. When TFE was added, the intensity of this peak decreased concomitantly with the appearance of minima around 205 nm and 222 nm whose intensity increased with the concentration of TFE. This is characteristic of an α-helical structure and the α-helical content of cementoin was estimated to be 48% in 50% TFE and up to 58% in 75% TFE. The observed isodichroic point at 203 nm indicates that the transition between the unstructured to the α-helical conformation is a two-state transition. Hence, a hydrophobic environment either induces or stabilizes α-helices in cementoin. This is in agreement with the AGADIR algorithm (Fig. 1D), which predicts the formation of two α-helices in cementoin: helix 1 with residues 10- 16 and helix 2 with residues 24-31, for a predicted total α-helical content of 39%.

Bottom Line: The N-terminal cementoin domain adopts α-helical secondary structures in a membrane mimetic environment, which is common in antimicrobial peptides.However, unlike numerous linear polycationic antimicrobial peptides, membrane disruption does not appear to be the main function of either cementoin, elafin or full-length pre-elafin/trappin-2 against P. aeruginosa.Our results rather suggest that pre-elafin/trappin-2 and elafin, but not cementoin, possess the ability to modulate the expression of some P.aeruginosa virulence factors, possibly through acting on intracellular targets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Biochimie, Microbiologie et Bio-informatique, Institut de Biologie Intégrative et des Systèmes and Regroupement PROTEO, Université Laval, Québec, Canada.

ABSTRACT

Background: Pre-elafin/trappin-2 is a human innate defense molecule initially described as a potent inhibitor of neutrophil elastase. The full-length protein as well as the N-terminal "cementoin" and C-terminal "elafin" domains were also shown to possess broad antimicrobial activity, namely against the opportunistic pathogen P. aeruginosa. The mode of action of these peptides has, however, yet to be fully elucidated. Both domains of pre-elafin/trappin-2 are polycationic, but only the structure of the elafin domain is currently known. The aim of the present study was to determine the secondary structures of the cementoin domain and to characterize the antibacterial properties of these peptides against P. aeruginosa.

Results: We show here that the cementoin domain adopts an α-helical conformation both by circular dichroism and nuclear magnetic resonance analyses in the presence of membrane mimetics, a characteristic shared with a large number of linear polycationic antimicrobial peptides. However, pre-elafin/trappin-2 and its domains display only weak lytic properties, as assessed by scanning electron micrography, outer and inner membrane depolarization studies with P. aeruginosa and leakage of liposome-entrapped calcein. Confocal microscopy of fluorescein-labeled pre-elafin/trappin-2 suggests that this protein possesses the ability to translocate across membranes. This correlates with the finding that pre-elafin/trappin-2 and elafin bind to DNA in vitro and attenuate the expression of some P. aeruginosa virulence factors, namely the biofilm formation and the secretion of pyoverdine.

Conclusions: The N-terminal cementoin domain adopts α-helical secondary structures in a membrane mimetic environment, which is common in antimicrobial peptides. However, unlike numerous linear polycationic antimicrobial peptides, membrane disruption does not appear to be the main function of either cementoin, elafin or full-length pre-elafin/trappin-2 against P. aeruginosa. Our results rather suggest that pre-elafin/trappin-2 and elafin, but not cementoin, possess the ability to modulate the expression of some P.aeruginosa virulence factors, possibly through acting on intracellular targets.

Show MeSH
Related in: MedlinePlus