Chemical approaches to unraveling the biology of mycobacteria

Abstract

Mycobacterium tuberculosis (Mtb), perhaps more than any other organism, is intrinsically appealing to chemical biologists. Not only does the cell envelope feature one of the most complex heteropolymers found in nature¹ but many of the interactions between Mtb and its primary host (we humans) rely on lipid and not protein mediators.^2,3 Many of the complex lipids, glycolipids, and carbohydrates biosynthesized by the bacterium still have unknown functions, and the complexity of the pathological processes by which tuberculosis (TB) disease progress offers many opportunities for these molecules to influence the human response. Because of the importance of TB in global public health, chemical biologists have applied a wide-ranging array of techniques to better understand the disease and improve interventions.

Figure 1.. A summary of small-molecule probes incorporated into Mtb cells.

(A) Universal activity-based probes (ABPs) have been produced that are based upon the inherent reactivity of fluorophosphonates towards esterases and ATP towards ATP-binding proteins. (B) Antibiotic-based ABPs have been produced that are based upon the inherent reactivity of THL and lalistat towards lipid hydrolases, carbapenems towards penicillin binding proteins, and EZ120P and BMB034 targeted serine hydrolases. (C) Fluorescent analogs of benzothiazinones, a novel class of covalent DprE1 inhibitors, have shown promise as whole cell labeling agents. (D) DDAO-OME and DCF-AME whose fluorescence is unmasked by serine esterases have been used for protein profiling of Mtb cells. Sal-AMS probe was designed to do protein profiling to study Sal-AMS inhibition in Mtb. (E) Several fluorogenic or luminogenic were reported for bacterial specific labeling, which are based on enzyme specificity of CDG series towards β-lactamase (and DprE1), FLASH towards Hip1 and DDAO-sulfate towards sulfatases. (F) Scanning electron micrograph of Mtb cells in mid log phase (false color applied).

Figure 2.. A summary of mycomembrane targeting probes incorporation into the Mtb cell envelope.

A wide variety of such probes have been incorporated into the mycomembrane of Mtb. Among the various strategies employed for probe incorporation, trehalose-based probes have been widely used. Trehalose in Mtb is found in the outer portion of the cell envelope as its corresponding mycolate lipids. Trehalose and its analogues are processed by Ag85 family enzymes and can be metabolically incorporated into outer membrane of Mtb for wider applications. (A) Cell wall schematic showing only the portions relevant for labeling and excludes many other molecules present in the mycobacterial outer membrane (B) Fluorescent probes based on modified trehalose e.g., FITC-Tre, FIT-Tre, DMN-Tre and 3HC-3-Tre. (C) azido-trehalose(s), fluoro-deoxy trehalose(s) and other alkyne functionalized trehalose probes for interrogation of the mycomembrane and detection of live Mtb in various samples, including potential positron emission tomography integrated with computed tomography (PET-CT) imaging agents and probes for protein profiling (D) fluorogenic probes provide the potential to turn-on after metabolism by Mtb i.e. FRET-TDM & QTF. (E and F) Probes based on unnatural d- amino acids and azido sugars have been widely applied to label mycobacterial peptidoglycan. (G) Bidentate boronic acids also bind to the lipoglycan components of mycomembrane.

Figure 3.. The utility of genetically encoded probes and advanced imaging in studying TB disease.

A. Both chemical and genetically encoded probes can be used simultaneously to probe the microenvironment of Mtb cells growing in various cells including various sub-compartments within macrophages. In the schematic two different phagosomes containing Mtb have been shown to be different by differential expression of environmentally sensitive protein fusions (for example, pH-responsive) combined with probes applied externally that are incorporated differentially (for example trehalose which is incorporated by replicating bacilli only). B. Use of real time positron emission tomography (PET) imaging using [¹⁸F]-fluorodeoxytrehalose to monitor disease progression in different mouse models of disease. C. [¹⁸F]-fluorodeoxyglucose PET-computed tomography (CT) scan of a TB patient showing the diversity of lesion types that are present. The feature at the top (red arrow) represents a cavity in the left apical region of the lung and the cartoon depicts the fibrotic wall of the cavity with some material remaining accompanied by an influx of neutrophils (blue cells with trilobed nuclei). The cartoon below shows an alternative lesion seen in this patient, a large granuloma with a caseous necrotic core. In both cartoons the Mtb bacilli are drawn in red and the blue, more spherical cells represent lymphocytes while the more elongated epitheloid cells are directly surrounding the necrotic core. Cavities are believed to result from necrosis of such lesions into the airways allowing rapid proliferation and dissemination.