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The self-assembly of a cyclic lipopeptides mixture secreted by a B. megaterium strain and its implications on activity against a sensitive Bacillus species.

Pueyo MT, Mutafci BA, Soto-Arriaza MA, Di Mascio P, Carmona-Ribeiro AM - PLoS ONE (2014)

Bottom Line: Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH.The aggregates are inactive per se at the pH of the culture medium which is around 6 or below.The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo SP, Brazil.

ABSTRACT
Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300-800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

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The CLP aggregates change their size with the environmental conditions such as ionic strength and pH but are not responsive to osmotic gradients evidencing their massive nature and the absence of an internal aqueous compartment.Effects of salt (a), osmotic gradient (b) and pH (c) on the kinetics of zeta-average diameter (Dz) of biosurfactant dispersions after adding different media. The effect of changing pH from 2 to 6 or to 8 on Dz and absorbance at 400 nm of the biosurfactant dispersion is shown in (d). Ethanol extracts of the CLP mixture (0.2 mL) were added of 3 mL water solutions (a, c) or 0.2 mL of ethanolic CLP were added of 4 mL of water solutions (b, d) forming a suspension of milky, opalescent appearance. The mixture was gently mixed until the water addition was complete.
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pone-0097261-g005: The CLP aggregates change their size with the environmental conditions such as ionic strength and pH but are not responsive to osmotic gradients evidencing their massive nature and the absence of an internal aqueous compartment.Effects of salt (a), osmotic gradient (b) and pH (c) on the kinetics of zeta-average diameter (Dz) of biosurfactant dispersions after adding different media. The effect of changing pH from 2 to 6 or to 8 on Dz and absorbance at 400 nm of the biosurfactant dispersion is shown in (d). Ethanol extracts of the CLP mixture (0.2 mL) were added of 3 mL water solutions (a, c) or 0.2 mL of ethanolic CLP were added of 4 mL of water solutions (b, d) forming a suspension of milky, opalescent appearance. The mixture was gently mixed until the water addition was complete.

Mentions: CLP ethanol extracts form turbid suspensions when mixed with Milli -Q water. Typically, adding the CLP ethanol extract to water solutions of monovalent salts (50 mM sodium salts) leads to an increase in particle size which could be determined by dynamic laser light scattering (Figure 5, panel a). Irrespective of the counterion ionic radius (Li+, K+, Na+), the ∼500 nm aggregates increased their diameter up to 1500 nm after 60 min. On panel b (Figure 5), the absence of osmotic response by CLP aggregates upon addition of a 100 mM D-glucose solution is shown. For closed vesicular structures, acting as semipermeable barriers, one should expect shrinkage and reduction in size induced by the hypertonic medium [29], [30]. On panel c (Figure 5), the CLP aggregation induced by a 50 mM NaCl solution is shown to depend on the pH being absent at acidic pH values such as 2.0. At pH∼7, when carboxyl and phosphate groups are deprotonated and negatively charged, the screening effect of 50 mM NaCl induces further CLP aggregation and increase in particle size (panel c on Figure 5). At pH∼2, the quoted groups are protonated and further aggregation upon addition of NaCl does not occur (panel c on Figure 5). To further assess the influence of pH on the aggregate size, a titration of CLP aggregates (starting at pH 2) with 50 mM NaOH was performed up to pH 8 as shown on the panel d of Figure 5. Both the turbidity at 400 nm and the mean particle size (Dz) remained relatively constant over a range of low pH values but over the 6–8 pH range, both turbidity and size of CLP aggregates abruptly decreased indicating the disaggregation of the CLP assemblies. Possibly, at the moderate ionic strength employed, the dissociation of negatively charged carboxylates and phosphates in the CLP molecules takes place over the 6–8 pH range inducing an increase in the intermolecular electrostatic repulsion and disassembly of the aggregates. Consistently, the antibiotic activity of the lyophilized CLP mixture solubilized in water against Bacillus cereus cells only occurs around pH 6–8 [12] when the CLP dispersions change from a turbid to a transparent and clear appearance in aqueous solution (panel d on Figure 5).


The self-assembly of a cyclic lipopeptides mixture secreted by a B. megaterium strain and its implications on activity against a sensitive Bacillus species.

Pueyo MT, Mutafci BA, Soto-Arriaza MA, Di Mascio P, Carmona-Ribeiro AM - PLoS ONE (2014)

The CLP aggregates change their size with the environmental conditions such as ionic strength and pH but are not responsive to osmotic gradients evidencing their massive nature and the absence of an internal aqueous compartment.Effects of salt (a), osmotic gradient (b) and pH (c) on the kinetics of zeta-average diameter (Dz) of biosurfactant dispersions after adding different media. The effect of changing pH from 2 to 6 or to 8 on Dz and absorbance at 400 nm of the biosurfactant dispersion is shown in (d). Ethanol extracts of the CLP mixture (0.2 mL) were added of 3 mL water solutions (a, c) or 0.2 mL of ethanolic CLP were added of 4 mL of water solutions (b, d) forming a suspension of milky, opalescent appearance. The mixture was gently mixed until the water addition was complete.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016289&req=5

pone-0097261-g005: The CLP aggregates change their size with the environmental conditions such as ionic strength and pH but are not responsive to osmotic gradients evidencing their massive nature and the absence of an internal aqueous compartment.Effects of salt (a), osmotic gradient (b) and pH (c) on the kinetics of zeta-average diameter (Dz) of biosurfactant dispersions after adding different media. The effect of changing pH from 2 to 6 or to 8 on Dz and absorbance at 400 nm of the biosurfactant dispersion is shown in (d). Ethanol extracts of the CLP mixture (0.2 mL) were added of 3 mL water solutions (a, c) or 0.2 mL of ethanolic CLP were added of 4 mL of water solutions (b, d) forming a suspension of milky, opalescent appearance. The mixture was gently mixed until the water addition was complete.
Mentions: CLP ethanol extracts form turbid suspensions when mixed with Milli -Q water. Typically, adding the CLP ethanol extract to water solutions of monovalent salts (50 mM sodium salts) leads to an increase in particle size which could be determined by dynamic laser light scattering (Figure 5, panel a). Irrespective of the counterion ionic radius (Li+, K+, Na+), the ∼500 nm aggregates increased their diameter up to 1500 nm after 60 min. On panel b (Figure 5), the absence of osmotic response by CLP aggregates upon addition of a 100 mM D-glucose solution is shown. For closed vesicular structures, acting as semipermeable barriers, one should expect shrinkage and reduction in size induced by the hypertonic medium [29], [30]. On panel c (Figure 5), the CLP aggregation induced by a 50 mM NaCl solution is shown to depend on the pH being absent at acidic pH values such as 2.0. At pH∼7, when carboxyl and phosphate groups are deprotonated and negatively charged, the screening effect of 50 mM NaCl induces further CLP aggregation and increase in particle size (panel c on Figure 5). At pH∼2, the quoted groups are protonated and further aggregation upon addition of NaCl does not occur (panel c on Figure 5). To further assess the influence of pH on the aggregate size, a titration of CLP aggregates (starting at pH 2) with 50 mM NaOH was performed up to pH 8 as shown on the panel d of Figure 5. Both the turbidity at 400 nm and the mean particle size (Dz) remained relatively constant over a range of low pH values but over the 6–8 pH range, both turbidity and size of CLP aggregates abruptly decreased indicating the disaggregation of the CLP assemblies. Possibly, at the moderate ionic strength employed, the dissociation of negatively charged carboxylates and phosphates in the CLP molecules takes place over the 6–8 pH range inducing an increase in the intermolecular electrostatic repulsion and disassembly of the aggregates. Consistently, the antibiotic activity of the lyophilized CLP mixture solubilized in water against Bacillus cereus cells only occurs around pH 6–8 [12] when the CLP dispersions change from a turbid to a transparent and clear appearance in aqueous solution (panel d on Figure 5).

Bottom Line: Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH.The aggregates are inactive per se at the pH of the culture medium which is around 6 or below.The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo SP, Brazil.

ABSTRACT
Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300-800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

Show MeSH
Related in: MedlinePlus