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Polycations increase the permeability of Mycobacterium vaccae cell envelopes to hydrophobic compounds
Małgorzata Korycka-Machała1, Andrzej Ziółkowski1, Anna Rumijowska-Galewicz1
1Centre for Microbiology & Virology, Polish Academy of Sciences, 93-232 Łódź, Lodowa 106, Poland1.
Abstract:
Polycations [protamine, polymyxin B nonapeptide (PMBN) and polyethyleneimine (PEI)] have been shown to increase the cell wall permeability of Mycobacterium vaccae to highly hydrophobic compounds, as manifested in enhanced intracellular bioconversion of beta-sitosterol to 4-androsten-3,17-dione (AD) and 1,4-androstadien-3,17-dione (ADD), and cell sensitization to erythromycin and rifampicin. The quantity of AD(D) formed per biomass unit was twice as high in the presence of PMBN and PEI, and three times higher with protamine. The sensitization factor, i.e. the MIC(50) ratio of the control bacteria to those exposed to polycations, ranged from 4 to 16, depending on the polycation/antibiotic combination. Non-covalently bound free lipids were extracted from the control and polycation-treated cells and fractionated with the use of chloroform, acetone and methanol. Chloroform- and acetone-eluted fractions (mainly neutral lipids and glycolipids, respectively) showed significant polycation-induced alterations in their quantitative and qualitative composition. The fatty acid profile of neutral lipids was reduced in comparison to control, whereas acetone-derived lipids were characterized by a much higher level of octadecenoic acid (C(18:1)) and a considerably lower content of docosanoic acid (C(22:0)), the marker compound of mycolate-containing glycolipids. Methanol-eluted fractions remained unaltered. Cell-wall-linked mycolates obtained from delipidated cells were apparently unaffected by the action of polycations, as judged from the TLC pattern of mycolic acid subclasses, the mean weight of mycolate preparations and the C(22:0) acid content in the mycolates, determined by GC/MS and pyrolysis GC. The results suggest the involvement of the components of non-covalently bound lipids in the outer layer in the M. vaccae permeability barrier.
Insights
Polycations like protamine, polymyxin B nonapeptide (PMBN), and polyethyleneimine (PEI) enhance Mycobacterium vaccae cell wall permeability. This increases hydrophobic compound bioconversion and antibiotic sensitivity, suggesting outer layer lipids are key to the permeability barrier.
Area of Science:
- Microbiology
- Biochemistry
- Cell Biology
Background:
- Mycobacterium vaccae possesses a cell wall that limits permeability to hydrophobic compounds.
- Polycations are known to interact with bacterial cell envelopes.
- Understanding permeability barriers is crucial for drug delivery and biotransformation processes.
Purpose of the Study:
- To investigate the effect of polycations (protamine, PMBN, PEI) on Mycobacterium vaccae cell wall permeability.
- To assess the impact of polycations on the bioconversion of beta-sitosterol and antibiotic susceptibility.
- To analyze alterations in the lipid composition of M. vaccae cells treated with polycations.
Main Methods:
- Treatment of M. vaccae with polycations (protamine, PMBN, PEI).
- Measurement of beta-sitosterol bioconversion to AD and ADD.
- Determination of antibiotic Minimum Inhibitory Concentration (MIC50) for erythromycin and rifampicin.
- Extraction and fractionation of non-covalently bound lipids using chloroform, acetone, and methanol.
- Analysis of lipid composition using Gas Chromatography-Mass Spectrometry (GC/MS) and Thin-Layer Chromatography (TLC).
Main Results:
- Polycations significantly increased the intracellular bioconversion of beta-sitosterol, with protamine showing a threefold increase and PMBN/PEI a twofold increase in AD(D) production.
- Polycation treatment sensitized M. vaccae to erythromycin and rifampicin, with sensitization factors ranging from 4 to 16.
- Alterations were observed in the quantitative and qualitative composition of chloroform- (neutral lipids) and acetone-eluted (glycolipids) fractions, notably changes in fatty acid profiles.
- Methanol-eluted fractions and cell-wall-linked mycolates remained largely unaffected by polycation treatment.
Conclusions:
- Polycations effectively enhance the permeability of the Mycobacterium vaccae cell wall to hydrophobic compounds and increase antibiotic sensitivity.
- The observed changes are primarily linked to alterations in the non-covalently bound lipids of the outer cell layer.
- These findings highlight the role of outer membrane lipids in M. vaccae's permeability barrier and suggest potential applications for polycations in bioprocessing and antimicrobial strategies.