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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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Scanning electron micrographs of P. aeruginosa incubated with cementoin, elafin, pre-elafin/trappin-2 or magainin 2. P. aeruginosa (~1 × 107 in 500 μL) were incubated 2 h with the indicated peptides before being processed for scanning electron microscopy as described in Methods. CNT; control performed in the absence of peptides, PE; pre-elafin/trappin-2, Cem; cementoin, Ela; elafin, Mag; magainin 2. White arrows point to pore-like structures and white arrowheads to ghost bacterial cells.
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Figure 2: Scanning electron micrographs of P. aeruginosa incubated with cementoin, elafin, pre-elafin/trappin-2 or magainin 2. P. aeruginosa (~1 × 107 in 500 μL) were incubated 2 h with the indicated peptides before being processed for scanning electron microscopy as described in Methods. CNT; control performed in the absence of peptides, PE; pre-elafin/trappin-2, Cem; cementoin, Ela; elafin, Mag; magainin 2. White arrows point to pore-like structures and white arrowheads to ghost bacterial cells.

Mentions: Positively charged α-helical peptides like cementoin, are characteristic of many AMPs. These were previously shown to either disrupt membranes and cause bacterial lysis or to translocate into the bacterial cytoplasm without causing cell lysis [19]. To obtain information about the mode of action of recombinant cementoin compared with that of elafin and pre-elafin/trappin-2 on P. aeruginosa, we first examined the effect of these peptides on bacteria by scanning electron micrography (SEM). As shown in Fig. 2, both elafin and cementoin significantly modified the appearance of P. aeruginosa cell surface with clear evidence of wrinkling, blister formation and the presence of pore-like structures (white arrows in Fig. 2). At the same concentration, pre-elafin/trappin-2 appeared to affect less severely the bacterial morphology and cells harboring pore-like structures were much less abundant. The presence of pores suggests that membrane integrity is compromised by addition of these peptides. However, ghost cells were rarely observed. In sharp contrast, when P. aeruginosa were exposed to magainin 2, a lytic AMP, much fewer cells could be visualized by SEM and ghost cells were numerous indicating cell lysis (white arrowheads in Fig. 2).


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)

Scanning electron micrographs of P. aeruginosa incubated with cementoin, elafin, pre-elafin/trappin-2 or magainin 2. P. aeruginosa (~1 × 107 in 500 μL) were incubated 2 h with the indicated peptides before being processed for scanning electron microscopy as described in Methods. CNT; control performed in the absence of peptides, PE; pre-elafin/trappin-2, Cem; cementoin, Ela; elafin, Mag; magainin 2. White arrows point to pore-like structures and white arrowheads to ghost bacterial cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Scanning electron micrographs of P. aeruginosa incubated with cementoin, elafin, pre-elafin/trappin-2 or magainin 2. P. aeruginosa (~1 × 107 in 500 μL) were incubated 2 h with the indicated peptides before being processed for scanning electron microscopy as described in Methods. CNT; control performed in the absence of peptides, PE; pre-elafin/trappin-2, Cem; cementoin, Ela; elafin, Mag; magainin 2. White arrows point to pore-like structures and white arrowheads to ghost bacterial cells.
Mentions: Positively charged α-helical peptides like cementoin, are characteristic of many AMPs. These were previously shown to either disrupt membranes and cause bacterial lysis or to translocate into the bacterial cytoplasm without causing cell lysis [19]. To obtain information about the mode of action of recombinant cementoin compared with that of elafin and pre-elafin/trappin-2 on P. aeruginosa, we first examined the effect of these peptides on bacteria by scanning electron micrography (SEM). As shown in Fig. 2, both elafin and cementoin significantly modified the appearance of P. aeruginosa cell surface with clear evidence of wrinkling, blister formation and the presence of pore-like structures (white arrows in Fig. 2). At the same concentration, pre-elafin/trappin-2 appeared to affect less severely the bacterial morphology and cells harboring pore-like structures were much less abundant. The presence of pores suggests that membrane integrity is compromised by addition of these peptides. However, ghost cells were rarely observed. In sharp contrast, when P. aeruginosa were exposed to magainin 2, a lytic AMP, much fewer cells could be visualized by SEM and ghost cells were numerous indicating cell lysis (white arrowheads in Fig. 2).

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