Download Mycolic acid export to the outer membrane of mycobacteria

NUS Graduate School for Integrative Sciences and Engineering
Research Project Write-up
Title of Project :
Mycolic acid export to the outer membrane of mycobacteria
Name of Supervisor :
Dr Chng Shu Sin
Contact Details:
[email protected]
Short Description
Our research program lies at the interface of chemistry and biology, and involves the
use of chemical, biochemical, genetic and biophysical approaches to characterize both the
chemistry and biology of a given system. The problem that my group is interested in studying
is membrane biogenesis, i.e. how a biological membrane is assembled. Membrane lipid
bilayers form the basis for life, physically defining cells and organelles, and modulating the
chemical environments within these compartments for optimal metabolism and growth.
Despite these fundamental roles, however, our understanding of membrane biogenesis has
remained rudimentary; we do not know how a cell makes more of itself. Conceptually, the
assembly of a membrane involves acquisition of its protein and lipid constituents from
another compartment where these molecules are synthesized. For lipids, this entails transport
from one membrane to another, usually across an aqueous environment. Our research group
is focused on using bacterial outer membranes as models to understand membrane biogenesis.
Specifically, we are interested in elucidating the mechanisms of inter-membrane lipid
trafficking in Gram-negative bacteria and mycobacteria.
The cell envelope of mycobacteria consists of two lipid bilayers: an inner (plasma)
membrane (IM) that encloses the aqueous cytoplasm and an outer membrane (OM) that faces
the extracellular milieu. Between these two membranes is a second aqueous compartment
known as the periplasm, which contains the cell wall that determines the shape of the bacterial
cell. This unique double-membrane envelope renders mycobacteria resistant to external
insults. The mycobacterial OM is a unique lipid bilayer whose detailed structure and
composition is not that well understood. The major component of the OM is mycolic acids,
which are C60-C90 long branched chain lipid moieties that covalently tether the OM to the
mycobacterial cell wall. These hydrophobic mycolic acids are believed to form the entire
inner leaflet of the mycobacterial OM, contributing to the low fluidity of the lipid bilayer and
thus rendering the OM an effective permeability barrier. Mycolic acids are bio-synthesized as
trehalose monomycolates (TMMs) in the IM, which are then transported across the IM,
periplasm and to the OM where they become functionalized onto the cell wall. The
biosynthesis of mycolic acids is quite well characterized. However, the final steps of TMM
synthesis and transport across the cell envelope remain unknown.
Mycobacterial infections, typified by tuberculosis (TB), are difficult to treat due to a
variety of reasons. One of these reasons is that many classes of antibiotics (macrolides,
glycopeptides, etc) are not effective because they cannot penetrate the OM. Furthermore,
resistance to effective drugs (isoniazid, rifampicin, etc) is already on the rise, underscoring the
need to invent new strategies to fight mycobacterial pathogens. Since the OM is essential for
the survival of these pathogens, and compromising OM integrity enables the use of many
antibiotics currently only effective against other pathogens that do not possess an OM, the
molecular machines that build or maintain the OM represent great targets for antibiotic
discovery. In particular, the biosynthetic pathway of mycolic acid, the major component of
the mycobacterial OM, is a well-validated target for first and second line TB drugs.
In this project, we focus our efforts on identifying proteins involved in TMM
biosynthesis and transport in Mycobacterium smegmatis and characterizing known systems
involved in these processes. Students in this project will be trained in areas such as basic
molecular biology and bacterial genetics, phenotypic assays, membrane fractionation, lipid
extraction/analysis, membrane protein expression/purification and protein-ligand interaction
analysis.