The power of solvatochromism : using environment-sensitive trehalose probes to detect mycobacteria with applications in research and medicine

Cell-surface trehalose mycolates are important modulators of mycobacterial pathogenesis and host immune response. Herein, we discuss the use of fluorescent and fluorogenic trehalose probes for the detection of the mycobacterial trehalose glycolipids with application in research and medicine. In Chapter 1, I review previous work leveraging the trehalose acylation pathway in mycobacteria to incorporate fluorescent reporters and/or affinity tags. In addition, I describe the utility of solvatochromic probes to report on the changes in hydrophobicity in the local environment. By combining these two previous unconnected concepts, I discuss a novel environment-sensitive probe, termed DMN-Tre, to probe mycobacterial cell wall. Lastly, I provide detailed recommended protocols for use of this reagent in laboratory and clinical settings. In Chapter 2, I offer a detailed analysis of the characteristics of DMN-Tre labeling in mycobacteria. DMN-Tre fluorescence undergoes > 700-fold increase in fluorescence intensity when transitioned from aqueous to hydrophobic environments. DMN-Tre labeling enabled the rapid, no-wash visualization of mycobacterial and corynebacterial species without nonspecific labeling of gram-positive or gram-negative bacteria. DMNTre labeling was detected within minutes and was inhibited by heat killing of mycobacteria. Furthermore, DMN-Tre labeling was reduced by treatment with TB drugs, unlike the clinically used auramine stain. Lastly, DMN-Tre labeled Mtb in TB-positive human sputum samples comparably to auramine staining, suggesting that this operationally simple method may be deployable for TB diagnosis. In Chapter 3, I explore a diverse set of solvatochromic dyes that may be brighter than DMN-Tre. We evaluated trehalose conjugates of 3-hydroxychromone (3HC) and Nile red (NR), alternative dyes that, like DMN, exhibit shifts in fluorescence wavelength and/or intensity in response to changes in environmental polarity. But unlike DMN, these dyes have extinction coefficients that are at least 5x higher and thus, they are much brighter dyes. One of the 3HC-trehalose probes, 3HC-3-Tre, demonstrated particularly favorable properties. Under no-wash conditions, 3HC-3-Tre labeled mycobacterial cells with 10- fold greater fluorescence intensity compared to DMN-Tre even when used at concentrations that were 10-fold lower. Consequently, due to the increase in brightness, we were able to detect Mtb cell labeling within 10 minutes of probe treatment. 3HC-3-Tre is therefore a promising reagent for the rapid detection of Mtb at the point of care. In Chapter 4, I investigate the practical usefulness of DMN-Tre or 3HC-3-Tre as diagnostic biomarkers in the clinical context. Excitingly, for the first time, we were able to detect intact and viable DMN-Tre -labeled Mtb cells in oral swabs from TB patient samples using an optimized protocol developed in this chapter. In addition, DMN-Tre was able to detect Mtb cells in blood samples from HIV/TB co-infected individuals. Importantly, DMN-Tre was able to monitor cell death of Mtb load in these samples during the course of a 72-hour treatment, suggesting that DMN-Tre is a promising reporter of treatment efficacy in real time. Lastly, in conjunction with a newly developed portable, low-cost and battery-powered fluorescent mini-microscope, we can rapidly detect mycobacteria using a simplified and automated process for the decentralized diagnosis of TB.

Keywords: Biology.
Advisor: Bertozzi Carolyn R.
Dixon Scott James
Stearns Tim