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Insights into the mode of action of the two-peptide lantibiotic haloduracin.

Oman TJ, van der Donk WA - ACS Chem. Biol. (2009)

Bottom Line: Despite significant structural differences between the two peptides of haloduracin and those of the two-peptide lantibiotic lacticin 3147, these two systems show similarities in their mode of action.Using Halalpha mutants, its B- and C-thioether rings are shown to be important but not required for bioactivity.A similar observation was made with mutants of Glu22, a residue that is highly conserved among several lipid II-binding lantibiotics such as mersacidin.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign and the Howard Hughes Medical Institute, Urbana, Illinois 61801, USA.

ABSTRACT
Haloduracin, a recently discovered two-peptide lantibiotic composed of the post-translationally modified peptides Halalpha and Halbeta, is shown to have high potency against a range of Gram-positive bacteria and to inhibit spore outgrowth of Bacillus anthracis. The two peptides display optimal activity in a 1:1 stoichiometry and have efficacy similar to that of the commercially used lantibiotic nisin. However, haloduracin is more stable at pH 7 than nisin. Despite significant structural differences between the two peptides of haloduracin and those of the two-peptide lantibiotic lacticin 3147, these two systems show similarities in their mode of action. Like Ltnalpha, Halalpha binds to a target on the surface of Gram-positive bacteria, and like Ltnbeta, the addition of Halbeta results in pore formation and potassium efflux. Using Halalpha mutants, its B- and C-thioether rings are shown to be important but not required for bioactivity. A similar observation was made with mutants of Glu22, a residue that is highly conserved among several lipid II-binding lantibiotics such as mersacidin.

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Potassium ion release from Micrococcus luteus cells induced by haloduracin and nisin. (a) Reponses to 10 (blue), 5 (green), 1 (red), 0.25 (orange), and 0 μM (gray) haloduracin or nisin. (b) The effects on potassium release by preincubating either Halα or Halβ prior to addition of its counterpeptide: (A, blue) 5 μM Halα preincubation followed by 5 μM Halβ addition; (B, green) 5 μM Halβ preincubation followed by 5 μM Halα addition; (C, red) simultaneous addition of 5 μM Halα and 5 μM Halβ; (D, orange) addition of 5 μM nisin; (E, violet) addition of 5 μM Halα; (F, pink) addition of 5 μM Halβ; and (G, gray) a control without haloduracin. Vertical line (black) indicates time of antibiotic addition.
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fig6: Potassium ion release from Micrococcus luteus cells induced by haloduracin and nisin. (a) Reponses to 10 (blue), 5 (green), 1 (red), 0.25 (orange), and 0 μM (gray) haloduracin or nisin. (b) The effects on potassium release by preincubating either Halα or Halβ prior to addition of its counterpeptide: (A, blue) 5 μM Halα preincubation followed by 5 μM Halβ addition; (B, green) 5 μM Halβ preincubation followed by 5 μM Halα addition; (C, red) simultaneous addition of 5 μM Halα and 5 μM Halβ; (D, orange) addition of 5 μM nisin; (E, violet) addition of 5 μM Halα; (F, pink) addition of 5 μM Halβ; and (G, gray) a control without haloduracin. Vertical line (black) indicates time of antibiotic addition.

Mentions: The effects of haloduracin on membrane potential prompted the investigation of potassium efflux using a cell impermeable, potassium-sensitive dye (PBFI). Treatment of a M. luteus cell suspension with Halα and Halβ triggered rapid efflux of intracellular potassium (6, panel a). Nearly identical results were observed for other Gram-positive strains such as Bacillus subtilis ATCC 6633 (data not shown). Potassium ion release was detected after a short lag time followed by a rapid signal increase that reached a plateau within 4−5 min. The observed potassium release was dose-dependent, and efflux was not detected in untreated samples. When cells were treated with Halα or Halβ alone at low micromolar concentrations, no leakage of potassium was observed. These findings, in addition to the results obtained from the sequential binding study described above, prompted us to further investigate the individual roles of the haloduracin peptides in pore formation. When Halα and Halβ were added together or when cells were preincubated with Halβ for 5 min followed by the addition of Halα, a short lag was observed for 20−30 s followed by rapid K+ efflux. However, when cells were preincubated with Halα followed by the addition of Halβ, rapid onset of efflux was observed and the lag was essentially absent. The combined results of potassium efflux and sequential binding assays suggest that the biological role of Halα involves binding to its target, resulting in a complex that serves as a docking site for Halβ to bind and form pores. A similar mechanism has been proposed for lacticin 3147 (9), and it may be that this is a general mechanism for two-peptide lantibiotics for which the α-peptide has structural homology with mersacidin.


Insights into the mode of action of the two-peptide lantibiotic haloduracin.

Oman TJ, van der Donk WA - ACS Chem. Biol. (2009)

Potassium ion release from Micrococcus luteus cells induced by haloduracin and nisin. (a) Reponses to 10 (blue), 5 (green), 1 (red), 0.25 (orange), and 0 μM (gray) haloduracin or nisin. (b) The effects on potassium release by preincubating either Halα or Halβ prior to addition of its counterpeptide: (A, blue) 5 μM Halα preincubation followed by 5 μM Halβ addition; (B, green) 5 μM Halβ preincubation followed by 5 μM Halα addition; (C, red) simultaneous addition of 5 μM Halα and 5 μM Halβ; (D, orange) addition of 5 μM nisin; (E, violet) addition of 5 μM Halα; (F, pink) addition of 5 μM Halβ; and (G, gray) a control without haloduracin. Vertical line (black) indicates time of antibiotic addition.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2812937&req=5

fig6: Potassium ion release from Micrococcus luteus cells induced by haloduracin and nisin. (a) Reponses to 10 (blue), 5 (green), 1 (red), 0.25 (orange), and 0 μM (gray) haloduracin or nisin. (b) The effects on potassium release by preincubating either Halα or Halβ prior to addition of its counterpeptide: (A, blue) 5 μM Halα preincubation followed by 5 μM Halβ addition; (B, green) 5 μM Halβ preincubation followed by 5 μM Halα addition; (C, red) simultaneous addition of 5 μM Halα and 5 μM Halβ; (D, orange) addition of 5 μM nisin; (E, violet) addition of 5 μM Halα; (F, pink) addition of 5 μM Halβ; and (G, gray) a control without haloduracin. Vertical line (black) indicates time of antibiotic addition.
Mentions: The effects of haloduracin on membrane potential prompted the investigation of potassium efflux using a cell impermeable, potassium-sensitive dye (PBFI). Treatment of a M. luteus cell suspension with Halα and Halβ triggered rapid efflux of intracellular potassium (6, panel a). Nearly identical results were observed for other Gram-positive strains such as Bacillus subtilis ATCC 6633 (data not shown). Potassium ion release was detected after a short lag time followed by a rapid signal increase that reached a plateau within 4−5 min. The observed potassium release was dose-dependent, and efflux was not detected in untreated samples. When cells were treated with Halα or Halβ alone at low micromolar concentrations, no leakage of potassium was observed. These findings, in addition to the results obtained from the sequential binding study described above, prompted us to further investigate the individual roles of the haloduracin peptides in pore formation. When Halα and Halβ were added together or when cells were preincubated with Halβ for 5 min followed by the addition of Halα, a short lag was observed for 20−30 s followed by rapid K+ efflux. However, when cells were preincubated with Halα followed by the addition of Halβ, rapid onset of efflux was observed and the lag was essentially absent. The combined results of potassium efflux and sequential binding assays suggest that the biological role of Halα involves binding to its target, resulting in a complex that serves as a docking site for Halβ to bind and form pores. A similar mechanism has been proposed for lacticin 3147 (9), and it may be that this is a general mechanism for two-peptide lantibiotics for which the α-peptide has structural homology with mersacidin.

Bottom Line: Despite significant structural differences between the two peptides of haloduracin and those of the two-peptide lantibiotic lacticin 3147, these two systems show similarities in their mode of action.Using Halalpha mutants, its B- and C-thioether rings are shown to be important but not required for bioactivity.A similar observation was made with mutants of Glu22, a residue that is highly conserved among several lipid II-binding lantibiotics such as mersacidin.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign and the Howard Hughes Medical Institute, Urbana, Illinois 61801, USA.

ABSTRACT
Haloduracin, a recently discovered two-peptide lantibiotic composed of the post-translationally modified peptides Halalpha and Halbeta, is shown to have high potency against a range of Gram-positive bacteria and to inhibit spore outgrowth of Bacillus anthracis. The two peptides display optimal activity in a 1:1 stoichiometry and have efficacy similar to that of the commercially used lantibiotic nisin. However, haloduracin is more stable at pH 7 than nisin. Despite significant structural differences between the two peptides of haloduracin and those of the two-peptide lantibiotic lacticin 3147, these two systems show similarities in their mode of action. Like Ltnalpha, Halalpha binds to a target on the surface of Gram-positive bacteria, and like Ltnbeta, the addition of Halbeta results in pore formation and potassium efflux. Using Halalpha mutants, its B- and C-thioether rings are shown to be important but not required for bioactivity. A similar observation was made with mutants of Glu22, a residue that is highly conserved among several lipid II-binding lantibiotics such as mersacidin.

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