Pathogenic species of mycobacteria and corynebacteria possess a thick and impermeable cell wall that helps these organisms evade detection and treatment by denying drug and probe molecules access to their cytoplasm. A distinctive feature of this barrier is an additional hydrophobic outer membrane composed of mycolic acids, which are covalently bound to an arabinogalactan (AG) polysaccharide that provides a link to the cell surface peptidoglycan. Efforts to identify weaknesses in these outer defenses would benefit greatly from a more complete understanding of how its components are assembled and maintained throughout the life cycle of the organisms. This project aims to develop chemical tools that specifically target and exploit the extracellular mycolyltransferase enzymes Ag85A-C, which mediate the covalent attachment of mycolic acids to the cell wall AG. First, we will detail a strategy to covalently label the cell wall mycolyl-arabinogalactan (mAG) complex with fluorophores to enable the visualization of mAG biosynthesis in real time. Our approach involves the design and synthesis of mimics of the endogenous mycolyl donor trehalose monomycolate (TMM), which carry fluorophore-conjugated mycolic acids. These substrates will be added to living bacteria, where we will evaluate their recognition by Ag85 and the incorporation of their fluorescent cargo into the growing cell wall. We anticipate that this robust labeling strategy will allow the localization and dynamics of cell wall assembly to be studied in unprecedented detail. Finally, we describe an approach to develop an Ag85-triggered turn-on fluorescent probe designed to fluoresce specifically in the presence of low concentrations of mycobacteria. We envisage that such a chemical tool would facilitate the early detection of pathogenic mycobacteria.

Public Health Relevance

The thick and impermeable cell wall of microorganisms like Mycobacterium tuberculosis and Corynebacterium diphtheriae is a formidable barrier to detecting and treating infection. We propose to exploit our knowledge of the enzymes that build this cell wall to devise chemical probes that can rapidly detect and label live microorganisms. These compounds are designed not only to detect pathogens but also to reveal how their cell wall changes during their life cycle.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM113454-01
Application #
8836071
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Barski, Oleg
Project Start
2015-02-15
Project End
2017-02-14
Budget Start
2015-02-15
Budget End
2016-02-14
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Hodges, Heather L; Brown, Robert A; Crooks, John A et al. (2018) Imaging mycobacterial growth and division with a fluorogenic probe. Proc Natl Acad Sci U S A 115:5271-5276