Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

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.

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