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Technologies for Emerging Infectious Diseases

Technologies for Emerging Infectious Diseases - Poster Session D

Poster K12 - Solvatochromic trehalose probes for detection of dormant mycobacteria

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Tuberculosis (TB) remains the most deadly infectious disease—briefly surpassed by the COVID-19 pandemic—and heavily impacts low-income countries with scarce resources for detection and treatment. Approximately 1.7 billion people harbor latent TB infections, caused by Mycobacterium tuberculosis (Mtb) in a dormant, non-replicative state. These latent infections can progress to active, life-threatening TB at any time. The global eradication of TB necessitates innovative tools for point-of-care detection of dormant Mtb. Current diagnostic methods, such as sputum smear microscopy with Ziehl-Neelsen or auramine staining, often fail to detect dormant mycobacteria due to reduced acid-fastness during dormancy, resulting in false negative results. To address this challenge, we previously reported on a novel solvatochromic probe, 4-N,N-dimethylamino-1,8-naphthalimide-conjugated trehalose (DMN-Tre), which fluoresces upon metabolic incorporation into the mycomembrane of live Mtb, catalyzed by antigen 85 (Ag85). Here, we demonstrate the efficacy of DMN-Tre in labeling both active and dormant mycobacteria, using low-carbon nutrient media to induce dormancy. We show that dormant bacteria remain viable and rapidly reactivate in nutrient-rich environments. DMN-Tre labeling intensity correlates with metabolic activity, making it a promising probe for investigating metabolic processes. DMN-Tre offers a low-cost, rapid, and specific method for detecting dormant Mtb and shows potential both as a clinical diagnostic for latent TB and as a scientific tool for understanding mycobacterial metabolism.

Materials and

Methods:  Preparation of active and dormant mycobacteria:
We cultured Mycobacterium smegmatis (Msmeg), a non-pathogenic model organism for Mtb, from an optical density (OD) of 0.1 in Middlebrook 7H9 media supplemented with oleic albumin dextrose catalase and glycerol (active) or unsupplemented (dormant). OD was measured on a microplate reader daily for 6 days.

Reactivation of dormant mycobacteria:
After 3 days, dormant Msmeg was resuspended in supplemented 7H9 to promote reactivation. Active Msmeg was resuspended in supplemented 7H9 as an active control, and dormant Msmeg in unsupplemented 7H9 as a dormant control.

ATP assays:
We performed ATP assays on active and dormant Msmeg over 6 days. Daily, samples were diluted to an OD of 0.1 and combined with BacTiter-Glo™ reagent (Promega, G8230). Assay luminescence was quantified on a microplate reader.

DMN-Tre labeling:
We labeled exponential-phase, stationary-phase, and dormant Msmeg with DMN-Tre after 3 and 6 days of incubation. Exponential-phase cultures were started 24 hours prior to labeling, while stationary-phase and dormant cultures were incubated for 6 days. For labeling, samples were diluted to an OD of 0.1 and incubated with 100 µM DMN-Tre for 30 minutes. Unlabeled control samples were incubated without DMN-Tre. Samples were washed twice in PBS, and fluorescence was quantified using flow cytometry (BD Accuri™ C6 Plus).

Auramine stain microscopy:
We stained exponential-phase, stationary-phase, and dormant Msmeg with auramine (ThermoFisher Scientific, R40094) after 4 days of incubation, and imaged stained and unstained samples with brightfield and fluorescence microscopy (EVOS M500, 40X objective, GFP channel).

Results, Conclusions, and Discussions: Our results confirm that carbon starvation induces dormancy in Msmeg. The reactivation of dormant cultures in nutrient-rich media demonstrates that bacteria remain viable throughout dormancy. Bacterial ATP concentrations in active cultures increase during the exponential phase and then decrease as cultures reach the stationary phase. In contrast, ATP concentrations in dormant cultures remain low, reflecting decreased metabolic activity.

Additionally, we find that DMN-Tre labels both active and dormant Msmeg. We observed significant mean fluorescence intensity in active and dormant labeled bacteria compared to unlabeled bacteria. Our findings demonstrate that, despite having low metabolic activity, non-replicating mycobacteria continue to metabolize trehalose. In active Msmeg, the fluorescence intensity of DMN-Tre labeling parallels metabolic activity: Fluorescence intensity decreases as cells approach the stationary phase.

Lastly, we performed fluorescence microscopy experiments to evaluate the potential of DMN-Tre and auramine for detecting latent TB. Preliminary microscopy results from auramine staining of active and dormant Msmeg confirm that acid-fast staining weakens during dormancy. While DMN-Tre labeling of active Msmeg varies with metabolic state, auramine staining remains consistent between exponential and stationary phases. These differences reflect the mechanisms of the two techniques—auramine stains existing components of the membrane, while DMN-Tre depends on metabolic formation and insertion of trehalose monomycolate. Results from these techniques suggest that dormant Msmeg exhibits altered membrane composition and maintains a low but significant rate of trehalose metabolism.

Future studies with DMN-Tre will include biochemical analyses, such as lipid extractions and enzyme inhibition assays, to further unravel the trehalose metabolic mechanisms in dormant bacteria.

The ability of DMN-Tre to label both active and dormant Msmeg demonstrates the probe’s potential for diagnosing latent TB. Moreover, DMN-Tre is a promising new tool for investigating changes in membrane composition and metabolism during mycobacterial dormancy. Future investigations using this probe may identify new targets for detection and treatment of latent TB infections.

Acknowledgements (Optional):