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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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Cyclic lipopeptides assemble as negatively charged aggregates with size distributions and mean sizes that change with sample processing exhibiting nevertheless very similar composition from MALDI-TOF mass spectra.Size distribution, mean hydrodynamic diameter (Dz) and zeta-potential from dynamic light scattering (a, b, c) and MALDI-TOF mass spectra for the B. megaterium culture supernatant (d, e, f) for the cell free, late stationary phase culture supernatant before (a, d) and after exhaustive dialysis against Milli-Q water (b, e) or after dialysis plus centrifugation at 17.000×g for 10 minutes (c, f).
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pone-0097261-g004: Cyclic lipopeptides assemble as negatively charged aggregates with size distributions and mean sizes that change with sample processing exhibiting nevertheless very similar composition from MALDI-TOF mass spectra.Size distribution, mean hydrodynamic diameter (Dz) and zeta-potential from dynamic light scattering (a, b, c) and MALDI-TOF mass spectra for the B. megaterium culture supernatant (d, e, f) for the cell free, late stationary phase culture supernatant before (a, d) and after exhaustive dialysis against Milli-Q water (b, e) or after dialysis plus centrifugation at 17.000×g for 10 minutes (c, f).

Mentions: In Figure 4, dynamic laser light scattering by fresh B. megaterium supernatants reveals particles having mean diameter up to ∼780 nm, a size consistent with large aggregates which were formed spontaneously in a mineral culture medium made up from salts, nitrogen and carbon sources (originally at pH∼8 but changing to lower values, ∼6–7, after growth to the late stationary phase). The aggregate size decreases after dialysis of the supernatant (compare panels a and b on Figure 4) as well as after dialysis and centrifugation at 17.000×g from ∼780 to 318 nm (compare panels a and c on Figure 4). The zeta potential, which measures the electrical potential at the shear plane in the particle neighborhood, is −46 mV (Figure 4a) and −48 mV (Figure 4c), barely changing after dialysis and centrifugation and showing a good colloidal stability for the negatively charged CLP aggregates. Figure 4 exhibits also MALDI-TOF MS of the dispersions both before (Figure 4d) and after dialysis against water or minimal medium (Figure 4e). They are qualitatively similar displaying the whole set of substances. After dialysis, all the peaks are retained by the dialysis membrane indicating aggregate formation. If after dialysis the suspension is centrifuged at 17.000×g, the supernatant still displays a similar composition (Figure 4f) suggesting aggregates small enough to remain in the supernatant after being centrifuged or alternatively, a set of disassembled substances at a concentration which is lower than the critical micelle concentration (CMC) and thus are not forming aggregates. The spontaneous assembly of CLP and low molecular weight lipids raises the question of what could be the role of such structures in the antibiotic activity. Likely, the aggregation concentrates active molecules in enormous particles in order to maximize the attack against the cell envelope. This inference may also be applied to other Bacillus species since they export a similar set of substances to the culture media (results not shown). Since the CLP mixture is secreted into a mineral culture medium having several inorganic salts and hence a relatively high ionic strength at the pH of water (pH 6.3), it is a reasonable assumption that from the CMC threshold, aggregates are formed. These, by effect of the ionic strength, could increase in size due not only to the screening of charges on the micelle ionic heads, but also due to the salting out effect on the hydrocarbon chains [28].


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)

Cyclic lipopeptides assemble as negatively charged aggregates with size distributions and mean sizes that change with sample processing exhibiting nevertheless very similar composition from MALDI-TOF mass spectra.Size distribution, mean hydrodynamic diameter (Dz) and zeta-potential from dynamic light scattering (a, b, c) and MALDI-TOF mass spectra for the B. megaterium culture supernatant (d, e, f) for the cell free, late stationary phase culture supernatant before (a, d) and after exhaustive dialysis against Milli-Q water (b, e) or after dialysis plus centrifugation at 17.000×g for 10 minutes (c, f).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0097261-g004: Cyclic lipopeptides assemble as negatively charged aggregates with size distributions and mean sizes that change with sample processing exhibiting nevertheless very similar composition from MALDI-TOF mass spectra.Size distribution, mean hydrodynamic diameter (Dz) and zeta-potential from dynamic light scattering (a, b, c) and MALDI-TOF mass spectra for the B. megaterium culture supernatant (d, e, f) for the cell free, late stationary phase culture supernatant before (a, d) and after exhaustive dialysis against Milli-Q water (b, e) or after dialysis plus centrifugation at 17.000×g for 10 minutes (c, f).
Mentions: In Figure 4, dynamic laser light scattering by fresh B. megaterium supernatants reveals particles having mean diameter up to ∼780 nm, a size consistent with large aggregates which were formed spontaneously in a mineral culture medium made up from salts, nitrogen and carbon sources (originally at pH∼8 but changing to lower values, ∼6–7, after growth to the late stationary phase). The aggregate size decreases after dialysis of the supernatant (compare panels a and b on Figure 4) as well as after dialysis and centrifugation at 17.000×g from ∼780 to 318 nm (compare panels a and c on Figure 4). The zeta potential, which measures the electrical potential at the shear plane in the particle neighborhood, is −46 mV (Figure 4a) and −48 mV (Figure 4c), barely changing after dialysis and centrifugation and showing a good colloidal stability for the negatively charged CLP aggregates. Figure 4 exhibits also MALDI-TOF MS of the dispersions both before (Figure 4d) and after dialysis against water or minimal medium (Figure 4e). They are qualitatively similar displaying the whole set of substances. After dialysis, all the peaks are retained by the dialysis membrane indicating aggregate formation. If after dialysis the suspension is centrifuged at 17.000×g, the supernatant still displays a similar composition (Figure 4f) suggesting aggregates small enough to remain in the supernatant after being centrifuged or alternatively, a set of disassembled substances at a concentration which is lower than the critical micelle concentration (CMC) and thus are not forming aggregates. The spontaneous assembly of CLP and low molecular weight lipids raises the question of what could be the role of such structures in the antibiotic activity. Likely, the aggregation concentrates active molecules in enormous particles in order to maximize the attack against the cell envelope. This inference may also be applied to other Bacillus species since they export a similar set of substances to the culture media (results not shown). Since the CLP mixture is secreted into a mineral culture medium having several inorganic salts and hence a relatively high ionic strength at the pH of water (pH 6.3), it is a reasonable assumption that from the CMC threshold, aggregates are formed. These, by effect of the ionic strength, could increase in size due not only to the screening of charges on the micelle ionic heads, but also due to the salting out effect on the hydrocarbon chains [28].

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