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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.

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Confocal microscopy of P. aeruginosa incubated with fluorescein-labeled pre-elafin/trappin-2. Mid-logarithmic phase cultures of P. aeruginosa were incubated for 1 h at 37°C with fluorescein-labeled pre-elafin/trappin-2 and observed by confocal microscopy at 400 × magnification. From left to right, consecutive 0.2 μm sections of a fluorescent bacterial cell.
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Figure 4: Confocal microscopy of P. aeruginosa incubated with fluorescein-labeled pre-elafin/trappin-2. Mid-logarithmic phase cultures of P. aeruginosa were incubated for 1 h at 37°C with fluorescein-labeled pre-elafin/trappin-2 and observed by confocal microscopy at 400 × magnification. From left to right, consecutive 0.2 μm sections of a fluorescent bacterial cell.

Mentions: Weak membrane depolarization and leakage of liposome-entrapped calcein, while indicating little membrane disruption, does not exclude that transient pores may form upon incubation of P. aeruginosa with pre-elafin/trappin-2 and derived peptides, as suggested by SEM examination. Formation of transient pores could lead to the translocation of the peptides across membranes. We previously reported that fluorescein-labeled pre-elafin/trappin-2 heavily decorated P. aeruginosa cells as assessed by fluorescence microscopy [27]. Here we used confocal microscopy to examine the fate of fluorescein-labeled pre-elafin/trappin-2 upon a 1 h incubation with P. aeruginosa. As shown in Fig. 4, the whole bacterial cell was fluorescent in all consecutive 0.2 μm sections. This is taken as evidence that pre-elafin/trappin-2 not only binds the surface, but also accumulates within the bacterial cytosol.


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)

Confocal microscopy of P. aeruginosa incubated with fluorescein-labeled pre-elafin/trappin-2. Mid-logarithmic phase cultures of P. aeruginosa were incubated for 1 h at 37°C with fluorescein-labeled pre-elafin/trappin-2 and observed by confocal microscopy at 400 × magnification. From left to right, consecutive 0.2 μm sections of a fluorescent bacterial cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Confocal microscopy of P. aeruginosa incubated with fluorescein-labeled pre-elafin/trappin-2. Mid-logarithmic phase cultures of P. aeruginosa were incubated for 1 h at 37°C with fluorescein-labeled pre-elafin/trappin-2 and observed by confocal microscopy at 400 × magnification. From left to right, consecutive 0.2 μm sections of a fluorescent bacterial cell.
Mentions: Weak membrane depolarization and leakage of liposome-entrapped calcein, while indicating little membrane disruption, does not exclude that transient pores may form upon incubation of P. aeruginosa with pre-elafin/trappin-2 and derived peptides, as suggested by SEM examination. Formation of transient pores could lead to the translocation of the peptides across membranes. We previously reported that fluorescein-labeled pre-elafin/trappin-2 heavily decorated P. aeruginosa cells as assessed by fluorescence microscopy [27]. Here we used confocal microscopy to examine the fate of fluorescein-labeled pre-elafin/trappin-2 upon a 1 h incubation with P. aeruginosa. As shown in Fig. 4, the whole bacterial cell was fluorescent in all consecutive 0.2 μm sections. This is taken as evidence that pre-elafin/trappin-2 not only binds the surface, but also accumulates within the bacterial cytosol.

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