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Polycations increase the permeability of Mycobacterium vaccae cell envelopes to hydrophobic compounds

Magorzata Korycka-Machaa¹

Andrzej Ziókowski¹

Centre for Microbiology & Virology, Polish Academy of Sciences, 93-232 ód, Lodowa 106, Poland¹
University of ód, Institute of Microbiology and Immunology, Department of Industrial Microbiology, 90-237 ód, Banacha 12/16, Poland²

Author for correspondence: Leon Sedlaczek. Tel: +48 42 6492263. Fax: +48 42 6491633. e-mail: lsedlacz{at}cmiwpan.lodz.pl

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 ß-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 MIC₅₀ 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.

Keywords: cell wall permeability, polycationic compounds, mycolates, free lipids

Abbreviations: AD, 4-androsten-3,17-dione; ADD, 1,4-androstadien-3,17-dione; F(M)AME, fatty (mycolic) acid methyl ester; OM, outer membrane; PEI, polyethyleneimine; PMBN, polymyxin B nonapeptide

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