J Biomol Struct Dyn. 2015;33(2):447-59.

Mobility and temperature resistance of trehalose mycolates as key characteristics of the outer membrane of Mycobacterium tuberculosis.

Migliardo F, Salmeron C, Bayan N.

Department of Physics and Earth Sciences , University of Messina , Viale D’Alcontres 31, 98166 Messina , Italy.



Trehalose mycolates are fundamental characteristics of the outer membrane (mycomembrane) of Mycobacterium tuberculosis and they are supposed to play a key role in the low permeability and high resistance of mycobacteria to many antibiotics; however, still, the molecular characteristics making mycolates so effective in their biological function are not fully understood. This work aims to investigate by quasi-elastic neutron scattering the diffusive dynamical properties of trehalose mycolates in water mixtures as a function of temperature, energy and exchanged wavevector Q in order to elucidate the dynamics-function relation in the mycomembrane. A comparison with lecithin lipids in water mixtures is performed since they are considered among the most rigid and resistant lipids. From the analysis of the data collected as a function of temperature, a lower temperature dependence of the mobility as well as a higher rigidity of trehalose mycolates in comparison with lecithin lipids are highlighted. The present findings provide detailed molecular information which allows to go ahead in the understanding at a molecular level of the resistance to stress and antibiotics by corynebacteria and mycobacteria.

KEYWORDS: Mycobacterium tuberculosis; diffusion; quasi elastic neutron scattering; stability; trehalose mycolates

PMID: 24601825


Additional text

Corynebacteriales, encompassing Mycobacteria, Corynebacteria, Rhodococcus and Nocardia, are Gram positive di-derm bacteria with a very specific outer lipid bilayer in their cell envelope. The presence of a hydrophobic barrier functionally related to the outer membrane of Gram-negative di-derm bacteria in these species, which is named mycomembrane (Figure 1), has been strongly suggested by electrophysiological and structural studies (1-4).


Figure 1 Cell envelope of Corynebacterium glutamicum


The biochemical analysis of the cell envelope of these bacteria revealed the presence of atypical lipids in the form of ester derivatives of mycolic acids, which include trehalose mono and dimycolates. Glycolipids and phospholipids are also present in the outer membrane. Bayan and co-workers (1,3,4) showed that the strong interaction between the mycolate outer membrane and the underlying polymer, mediated though esterification of mycolates to the arabinose of the arabinogalactane polymer, is essential for cell envelope integrity.

The cell envelope of mycobacteria and corynebacteria represent a very efficient impermeable barrier that contributes to their high resistance to hydrophilic drugs and is essential for their virulence (5-8). Several studies emphasize the responsibility of mycolate layer for the human pathogen Mycobacterium tuberculosis low permeability, persistence in the host cell and resistance to the commonly used antibiotics.


Figure 2 Isolation of purified outer mycolate membrane from a C. glutamicum ∆aftB strain


The present research moves from the observation that free trehalose plays a significant bioprotective role for several organisms under stress conditions (9-11). The spectroscopic data obtained by complementary techniques (12-15) points out that trehalose is capable to remarkably modify the both the water structural and dynamical properties by a strong interaction which leads to the creation of a rigid environment where biomolecules can be protected. The specific goal of our work is to understand if trehalose exerts the cryptobiotic functions shown when it is free also when it is bound in mycolates. Due to the many biological functions of trehalose mycolates, such a study could impact on survival of mycobacteria within the host and possibly their virulence.

In order to achieve this objective, we performed a combined biochemical and biophysical study.

The research has been carried out on C. glutamicum ATCC 13032 reference strain, a recognized model for Mycobacterium tuberculosis cell envelope studies. Outer membrane vesicles were purified from 300 ml ΔaftB BHI cultures, a mutant strain whose outer mycomembrane is destabilized (see Figure 2, left panel) and shed in the culture medium. The bacteria were cultured in liquid brain heart infusion (BHI) medium at 30°C and centrifuged at 200 rpm for 24 h. The supernatant was submitted to ultracentrifugation and the resulting pellet purified on a sucrose gradient centrifugation (see Figure 2 right panel). The band corresponding to the outer membrane was collected, washed twice to remove sucrose, resuspended in the HEPES buffer and observed by freeze fracture electron microscopy (see Figure 2). The fraction has also been biochemically analyzed by thin-layer chromatography (TLC) after lipid extraction in appropriate solvants (3). The lipids were detected by spraying plates with 0.2% anthrone in concentrated H2SO4, followed by heating. TLC revealed two main dark-blue spots that can be attributed to trehalose dimycolates (TDM) and trehalose monomycolates (TMM). Only small traces of phospholipids were presents as minor spots after specific revelation (not shown). The ratio between TDM and TMM is about 1 (see Figure 3).



Figure 3 TLC analysis of purified outer mycolate membrane from C. glutamicum


The diffusive dynamical properties of mixtures of the trehalose monomycolates and dimycolates (extracted from purified outer membrane as described before) in the ratio of 1:1 and at a concentration value of 300 mg/ml both in H2O and in D2O have been investigated for the first time by Elastic (ENS) and Quasi Elastic Neutron Scattering (QENS) experiments performed as a function of temperature and exchanged vector Q by using the IRIS and OSIRIS spectrometers at the ISIS facility (RAL, UK) (16-17). A comparison between the diffusive dynamics of trehalose-containing lipids, i. e. trehalose mycolates, and that of trehalose-lacking, i. e. lecithin lipids, has been performed to elucidate the role of the disaccharide in the observed differences.

The results show that the dependence on temperature and Q of the QENS spectra and of the derived quantities, such as the translational linewidths and EISF, is weaker for trehalose mycolates than for lecithin lipids, so pointing out a lower mobility, a lower flexibility and higher resistance of mycolates to the thermal stress. It is possible to conclude that trehalose plays a key-role in conferring the high impermeability and rigidity properties to the outer membrane of corynebacteria and mycobacteria, which are responsible for the bacterial response to stress and antibiotics.


Figure 4 Translational linewidths and EISF for trehalose mycolates and lecithin lipids.



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The authors gratefully acknowledge the ISIS facility (STFC, RAL, Chilton, UK) for dedicated runs at the OSIRIS and IRIS spectrometers. Federica Migliardo gratefully acknowledges the European Molecular Biology Organisation (EMBO) for the Short-term Fellowship “Role of trehalose in the structure, permeability and stability of mycomembranes in Mycobacterium tuberculosis and Corynebacterium glutamicum” [ASTF 502 – 2011].



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