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Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts
95
32
-POS
Board 32
Small Hydrophilic Molecule Permeation in Mycobacterium Tuberculosis
Raju Mukherjee
1
, Thomas Ioerger
2
, Digby F. Warner
1
, Valerie Mizrahi
1
.
1
Institute of infectious disease and molecular medicine, Cape Town, South Africa,
2
Texas A&M
university, College Station, TX, USA.
Despite originating from different chemical classes, “new chemical entities” identified by
screening for compounds with whole-cell activity against Mycobacterium tuberculosis share a
number of features: they are relatively hydrophobic, and frequently appear to target a small set of
“promiscuous” targets – specifically, DprE1, MmpL3, Pks13 and QcrB – which are either
located in the periplasm, or are associated with the mycobacterial plasma (inner) membrane. The
relative paucity of hydrophilic compounds identified by phenotypic screening is thought to be
attributable, at least in part, to the formidable permeability barrier presented by the complex,
lipid-rich mycobacterial cell wall, which comprises the mycobacterial outer membrane
(mycomembrane), periplasmic space, and plasma membrane. Discovery-driven mass
spectrometry (MS), also known as “shotgun MS”, has been widely used for qualitative and
quantitative measurements of proteomes. In order to identify outer membrane proteins that may
be involved in the uptake of small hydrophilic molecules, including a number of standard anti-
TB drugs, we have employed a shotgun MS approach to the mycomembrane sub-proteome of M.
tuberculosis. Proteins from the mycomembrane of logarithmic and stationary-phase M.
tuberculosis cells were isolated by detergent extraction using the non-ionic detergent, n-octyl-β-
D-glucoside, and Triton X-100. Successive tryptic digestion and MS-based searches resulted in
the identification of a number of hypothetical and conserved hypothetical proteins. In parallel,
we have also employed high density transposon mutagenesis coupled with deep sequencing (Tn-
Seq) to identify the conditionally essential genes under the specific growth condition and hence
can be implicated for uptake of small molecules including inorganic nitrate. The identification
and preliminary functional characterization of a subset of these proteins is described here.