Unsupported planar lipid membranes formed from mycolic acids of Mycobacterium tuberculosis (original) (raw)
Related papers
Biophysical Journal, 2008
Mycobacteria, including persistent pathogens like Mycobacterium tuberculosis, have an unusual membrane structure in which, outside the plasma membrane, a nonfluid hydrophobic fatty acid layer supports a fluid monolayer rich in glycolipids such as trehalose 6,69-dimycolate (TDM; cord factor). Given the abilities of mycobacteria to survive desiccation and trehalose in solution to protect biomolecules and whole organisms during freezing, drying, and other stresses, we hypothesized that TDM alone may suffice to confer dehydration resistance to the membranes of which it is a constituent. We devised an experimental model that mimics the structure of mycobacterial envelopes in which an immobile hydrophobic layer supports a TDM-rich, two-dimensionally fluid leaflet. We have found that TDM monolayers, in stark contrast to phospholipid membranes, can be dehydrated and rehydrated without loss of integrity, as assessed by fluidity and protein binding. Strikingly, this protection from dehydration extends to TDM-phospholipid mixtures with as little as 25 mol % TDM. The dependence of the recovery of membrane mobility upon rehydration on TDM fraction shows a functional form indicative of spatial percolation, implying that the connectivity of TDM plays a crucial role in membrane preservation. Our observations are the first reported instance of dehydration resistance provided by a membrane glycolipid.
Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane
Nature Chemical Biology, 2012
New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Permeation Across the Mycomembrane in Live Mycobacteria
bioRxiv (Cold Spring Harbor Laboratory), 2022
The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most commonly ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. Nonetheless, there is limited information on the types of molecular scaffolds that can readily permeate past the mycomembrane of mycobacteria. To address this, we describe a novel assay that combines metabolic tagging of the peptidoglycan scaffold, which sits directly beneath the mycomembrane, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in Mycobacterium smegmatis and Mtb. A small panel of molecules was tested and we found a large range in the permeability profile of molecules. Interestingly, the general trend is similar across the two types of mycobacteria, with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria. The methods described, which do not require genetic manipulation, can be generally adopted to other species for which envelope permeability is treatment-limiting.
Progress in Lipid Research, 2012
Mycolic acids constitute the waxy layer of the outer cell wall of Mycobacterium spp. and a few other genera. They are diverse in structure, providing a unique chromatographic foot-print for almost each of the more than 70 Mycobacterium species. Although mainly esterified to cell wall arabinogalactan, trehalose or glucose, some free mycolic acid is secreted during in vitro growth of Mycobacterium tuberculosis. In M.
Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State
Journal of Bacteriology, 2008
The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.
Biochemistry and molecular genetics of cell-wall lipid biosynthesis in mycobacteria
Molecular Microbiology, 1997
Tuberculosis and other mycobacterial infections are the most serious infectious diseases in terms of human fatalities. The high content of unique cellwall lipids helps these organisms to resist antimicrobial drugs and host defences. The biosynthesis of these lipids is discussed briefly. The recent advances in recombinant DNA technology have begun to help to elucidate the nature of some of the enzymes involved in this process and the genes that encode them. Gene disruption and other molecular genetic technologies are beginning to provide new approaches to test for the biological functions of these gene products and may lead to identification of new antimycobacterial drug targets.
The Mycobacterial Membrane: A Novel Target Space for Anti-tubercular Drugs
Frontiers in microbiology, 2018
Tuberculosis (TB) poses an enduring threat to global health. Consistently ranked among the top 10 causes of death worldwide since 2000, TB has now exceeded HIV-AIDS in terms of deaths inflicted by a single infectious agent. In spite of recently declining TB incident rates, these decreases have been incremental and fall short of threshold levels required to end the global TB epidemic. As in other infectious diseases, the emergence of resistant organisms poses a major impediment to effective TB control. Resistance in mycobacteria may evolve from genetic mutations in target genes which are transmitted during cell multiplication from mother cells to their progeny. A more insidious form of resistance involves sub-populations of non-growing ("dormant") mycobacterial persisters. Quiescent and genetically identical to their susceptible counterparts, persisters exhibit non-inheritable drug tolerance. Their prevalence account for the protracted treatment period that is required for ...
Differential spontaneous folding of mycolic acids from Mycobacterium tuberculosis
Chemistry and Physics of Lipids Volume 180, May 2014, Pages 15–22
Mycolic acids are structural components of the mycobacterial cell wall that have been implicated in the pathogenicity and drug resistance of certain mycobacterial species. They also offer potential in areas such as rapid serodiagnosis of human and animal tuberculosis. It is increasingly recognized that conformational behavior of mycolic acids is very important in understanding all aspects of their function. Atomistic molecular dynamics simulations, in vacuo, of stereochemically defined Mycobacterium tuberculosis mycolic acids show that they fold spontaneously into reproducible conformational groupings. One of the three characteristic mycolate types, the keto-mycolic acids, behaves very differently from either α-mycolic acids or methoxy-mycolic acids, suggesting a distinct biological role. However, subtle conformational behavioral differences between all the three mycolic acid types indicate that cooperative interplay of individual mycolic acids may be important in the biophysical properties of the mycobacterial cell envelope and therefore in pathogenicity.
Topology of the Porin MspA in the Outer Membrane of Mycobacterium smegmatis
Journal of Biological Chemistry, 2005
MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic -barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surfaceexposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic -barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nmlong part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic ␣-helices or -sheets are integrated into a lipid membrane.