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Understanding the organisation of the spatially and compositionally exclusive lipids of the mycobacterial inner and outer membranes

Understanding the organisation of the spatially and compositionally exclusive lipids of the mycobacterial inner and outer membranes 

Introduction

Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens and causes tuberculosis – one of the leading causes of death worldwide. Mtb is one of the most robust bacteria present and this is attributed to its remarkable defence mechanism and the use of the same as offense to survive the host inflammatory response. This is the bacteria’s complex cell envelope architecture and the lipids associated with it which is common to Mycobacteriaceae family. The bacteria employ long chained (C60-90) atypical lipids that have extensive branching to make up the cell envelope. The location of each of these long chains lipids are distinct form one and other in terms of their spatial arrangement i.e the inner membrane (IM) and the outer membrane (OM) of the bacteria. The inner membrane consists of the conventional phospholipids and phosphatidyl mannosides. The cell wall now covers this inner membrane with a complex mesh of peptidoglycan-arabinogalactan matrix. This is in turn covalently linked to what is the beginning of the outer membrane of the bacteria via long chain mycolic acids (MA) that are about C60-90 in length. The outer membrane consists of atypical lipids that are long chained, highly branched and non-covalently localized like trehalose dimycolate (TDM), sulfolipids (SLs) and more. In this work, we extract and reconstitute the membrane lipids in combinations that would describe only the OM, the IM and a system that would closely resemble the whole cell envelope (Mycomembrane). We then report the membrane properties of these distinct lipid bilayers and their functional significance using a surrogate model (Mycobacterium smegmatis (Msm)), a commonly used species for studying the Mycobacteriaceae family. These two species share a high percentage of their lipidome with only variations in about 20-25% of the lipid population which could be attributed to the virulence of Mtb and not of Msm.

Novelty

Mycobacterial membranes pose a challenge when it comes to the permeability of drugs across its membrane and the development of new antimycobacterial drugs. This work would help lay the foundation to understand and investigate the advantages conferred on the bacteria by these lipids, the functional roles of these lipids in drug resistance, and also help in the designing of membrane active compounds specific to mycobacterial membranes.  It would also help serve as a mycobacteria specific model for the screening of drugs and the efficiency of antimycobacterial drug delivery systems. 

Methodologies adopted

The lipids form the bacteria were first extracted to obtain each of the layers of the cell envelope without any cross contamination from the others using traditional separation techniques based on solubility and polarity of each of the lipid bilayers. In this work we employ the use of spectroscopy – Fluorescence and Infrared, to understand the membrane packing, hydration and fluidity of the bilayers. Atomic force microscopy and Fluorescence confocal microscopy give us a visual insight into the membrane topology and the arrangement of the membrane domains. 

Most significant results

The outer membrane lipids are more loosely packed and show higher fluctuations associated with higher interfacial hydration. The inner membrane on the other hand due to the presence of more saturated lipids showed lower levels of hydration and therefore a tighter packing. When we combine the systems to resemble the cell envelope closely, by incorporating the MA and the glycan associated lipids, we now see an interesting lipid-lipid interaction that now fine tunes the ordering of all the membranes together to a more ordered state negating the effects of the OM. Therefore we see a lipid order gradient that is now spatially established starting from the outer membrane and gradually increasing towards the inner membrane of the bacteria. 

Conclusion

Understanding the organization of these lipids, accentuates our ideas on of how the bacteria forms this robust cell envelope and guards itself. This gradient of membrane order and fluidity suggests an arrangement to regulate the uptake of nutrients and the differential interaction with drugs. Although our results are obtained from protein free systems, the findings in this work will help in future studies targeted towards understanding and uncovering new potential targets against mycobacterial infections, tuberculosis in specific and also our model could help in aiding the development of drug delivery systems against the virulent Mycobacteriaceae family members.