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Lipid domains of mycobacteria studied with fluorescent molecular probes
H Christensen1, N J Garton, R W Horobin
1Department of Microbiology, Medical School, University of Newcastle, Newcastle upon Tyne, UK.
Abstract:
The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non-tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non-covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl-arabinogalactan-peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high-resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.
Insights
Researchers visualized native lipid organization in mycobacterial cell envelopes using fluorescent probes. This reveals distinct lipid domains and the structure of the mycolyl-arabinogalactan-peptidoglycan complex, crucial for understanding these pathogens.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- The mycobacterial cell envelope is key to pathogenesis but its lipid organization is poorly understood.
- Conventional methods disrupt the native distribution of non-covalently bound lipids.
Purpose of the Study:
- To investigate the native organization of lipid domains within the mycobacterial cell envelope.
- To visualize the structure of the mycobacterial envelope using advanced imaging techniques.
Main Methods:
- Application of fluorescent lipophilic probes to live mycobacteria (M. smegmatis, M. tuberculosis, M. avium, M. gadium, M. aurum).
- Analysis using high-resolution fluorescence microscopy and digital image processing.
- Utilized fluorescence quenching, detergents, and solvents to probe lipid domain structure.
Main Results:
- Identified distinct intracellular and envelope lipid domains.
- Lipid probe localization correlated with hydrophobicity and amphiphilicity.
- Observed uneven lipid distribution and selective labeling of septal regions.
- Demonstrated a coherent cell envelope lipid domain and directly imaged the mycolyl-arabinogalactan-peptidoglycan complex.
Conclusions:
- Fluorescent probes and microscopy provide a clear view of distinct mycobacterial lipid domains.
- Understanding lipid organization is vital for deciphering mycobacterial physiology and developing new treatments.
- This approach offers new insights into the structure of the mycobacterial cell envelope.