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Honey glycoproteins containing antimicrobial peptides, Jelleins of the Major Royal Jelly Protein 1, are responsible for the cell wall lytic and bactericidal activities of honey.

Brudzynski K, Sjaarda C - PLoS ONE (2015)

Bottom Line: Using Concavalin A-affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties.The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively.Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery and Development Department, Bee-Biomedicals Inc., St. Catharines, Ontario, Canada.

ABSTRACT
We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A-affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey's active principle responsible for this activity.

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Glycoprotein- induced cell wall damage and cell shape changes in log-phase E. coli and B. subtilis cultures.A. E. coli. a. untreated control, b. E. coli incubated with G177, c. G208 and d. G210. B. B. subtilis. a. untreated control, b. B. subtilis incubated with G177, c. G208 and d. G210.
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pone.0120238.g004: Glycoprotein- induced cell wall damage and cell shape changes in log-phase E. coli and B. subtilis cultures.A. E. coli. a. untreated control, b. E. coli incubated with G177, c. G208 and d. G210. B. B. subtilis. a. untreated control, b. B. subtilis incubated with G177, c. G208 and d. G210.

Mentions: Exposure of overnight bacterial cultures to glps resulted in visible agglutination of both Gram- positive Bacillus subtilis (ATCC-6633) and Gram-negative Escherichia coli (ATCC-14948) (Fig. 3). All four glycoproteins clumped bacterial cells in a similar way to ConA-lectin as compared to soybean lectin (Fig. 3a and b). To investigate whether, in addition to agglutinating activity, glps have capacity to influence the growth and bacterial survival, overnight cultures of B. subtilis and E. coli (diluted to 106 CFU/ml) were incubated with serially two-fold diluted glps for 18 h. At two time-points of bacterial growth, at the beginning of log-phase and at the stationary phase, 10μl aliquots were withdrawn from incubation wells for microscopic observations. The microscopic images revealed marked morphological changes that included filamentation, cell wall lysis and formations of spheroplasts (Figs. 4 and 5). The extensive disruption of the bacterial cell walls occurred indiscriminately in both Gram-positive B. subtilis and Gram-negative and E. coli (Fig. 4A and B). As a result, at the entry to a stationary phase, the majority of E. coli and B. subtilis cells had their cell-shape changed from the rod-like forms to spheroidal forms of different sizes (Fig. 5).


Honey glycoproteins containing antimicrobial peptides, Jelleins of the Major Royal Jelly Protein 1, are responsible for the cell wall lytic and bactericidal activities of honey.

Brudzynski K, Sjaarda C - PLoS ONE (2015)

Glycoprotein- induced cell wall damage and cell shape changes in log-phase E. coli and B. subtilis cultures.A. E. coli. a. untreated control, b. E. coli incubated with G177, c. G208 and d. G210. B. B. subtilis. a. untreated control, b. B. subtilis incubated with G177, c. G208 and d. G210.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4382210&req=5

pone.0120238.g004: Glycoprotein- induced cell wall damage and cell shape changes in log-phase E. coli and B. subtilis cultures.A. E. coli. a. untreated control, b. E. coli incubated with G177, c. G208 and d. G210. B. B. subtilis. a. untreated control, b. B. subtilis incubated with G177, c. G208 and d. G210.
Mentions: Exposure of overnight bacterial cultures to glps resulted in visible agglutination of both Gram- positive Bacillus subtilis (ATCC-6633) and Gram-negative Escherichia coli (ATCC-14948) (Fig. 3). All four glycoproteins clumped bacterial cells in a similar way to ConA-lectin as compared to soybean lectin (Fig. 3a and b). To investigate whether, in addition to agglutinating activity, glps have capacity to influence the growth and bacterial survival, overnight cultures of B. subtilis and E. coli (diluted to 106 CFU/ml) were incubated with serially two-fold diluted glps for 18 h. At two time-points of bacterial growth, at the beginning of log-phase and at the stationary phase, 10μl aliquots were withdrawn from incubation wells for microscopic observations. The microscopic images revealed marked morphological changes that included filamentation, cell wall lysis and formations of spheroplasts (Figs. 4 and 5). The extensive disruption of the bacterial cell walls occurred indiscriminately in both Gram-positive B. subtilis and Gram-negative and E. coli (Fig. 4A and B). As a result, at the entry to a stationary phase, the majority of E. coli and B. subtilis cells had their cell-shape changed from the rod-like forms to spheroidal forms of different sizes (Fig. 5).

Bottom Line: Using Concavalin A-affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties.The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively.Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery and Development Department, Bee-Biomedicals Inc., St. Catharines, Ontario, Canada.

ABSTRACT
We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A-affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey's active principle responsible for this activity.

Show MeSH
Related in: MedlinePlus