TowardsMycobacterium tuberculosisdetection at the point-of-care: a brighter solvatochromic probe permits the detection of mycobacteria within minutes

Abstract

25There is an urgent need for point-of-care tuberculosis (TB) diagnostic methods that are fast, 26 inexpensive, and operationally simple. Here, we report on a bright solvatochromic dye trehalose 27 conjugate that specifically detects Mycobacterium tuberculosis (Mtb) in minutes.3-28 hydroxychromone (3HC) dyes, known to yield high fluorescence quantum yields, exhibit shifts in 29 fluorescence intensity in response to changes in environmental polarity. We synthesized two 30 analogs of 3HC-trehalose conjugates (3HC-2-Tre and 3HC-3-Tre) and determined that 3HC-3- 31 Tre has exceptionally favorable properties for Mtb detection. 3HC-3-Tre-labeled mycobacterial 32 cells displayed a 10-fold increase in fluorescence intensity compared to our previously reports on 33 the dye 4,4-N,N-dimethylaminonapthalimide (DMN-Tre). Excitingly, we detected fluorescent Mtb 34 cells within 10 minutes of probe treatment. Thus, 3HC-3-Tre permits rapid visualization of 35 mycobacteria that ultimately could empower improved Mtb detection at the point-of-care in low-36 resource settings. 37 38 With 1.2 million deaths and 10 million new cases in 2018, tuberculosis (TB) is the most 39 lethal infectious disease in the world (1). Early detection of the bacterium Mycobacterium 40 tuberculosis (Mtb), the causative agent of TB, followed by appropriate treatment could prevent 41 most deaths (2). The gold standard for TB diagnosis remains a labor-intensive culture test that 42 requires weeks of incubation time in specialized facilities. Although more rapid tests are available, 43 they present several important limitations. PCR-based tests are expensive and require skilled 44 technicians. Microscopy-based methods are attractive in low-resource settings as they are low-45 cost, have fast turnaround times, and report on people at greatest risk of transmission and death 46 (3). As a result, the sputum smear microscopy test is the most widely used technique for TB 47 diagnosis. The century-old smear test is based on the susceptibility of fluorescent auramine dye 48 or colored Ziehl-Neelson (ZN) stain to accumulate within the highly hydrophobic mycobacterial 49 cell wall (4-7). While effective for identification of Mtb cells, this process requires multiple wash 50 steps to reduce non-specific background fluorescence, or in the case of the ZN test, a rigorous 51counterstaining procedure so that stained Mtb cells can be visualized (8,9). Moreover, the smear 52 test does not distinguish live from dead cells, and this capability is vital in order to assess 53 treatment efficacy early and accurately (2). 54In the last decade, we and others have leveraged the trehalose metabolism of 55 mycobacteria to mark them for detection by various imaging methods (10-20). Exogenous 56 trehalose molecules can be directly mycolylated at the 6 position by antigen 85 (Ag85) enzymes 57 to form trehalose monomycolates (TMM) that are inserted into the mycobacterial cell wall, termed 58 the mycomembrane (10). Researchers have shown that Ag85 enzymes are promiscuous enough 59 to ...

Metabolic labeling probes for interrogation of the host–pathogen interaction

“…This allowed the detection of Mtb cells within 10 minutes of treatment and may well represent a major step towards on-site testing for Mtb infection. 111 Hodges et al recently reported the use of a different strategy for fluorogenic Mtb cell wall detection. Their approach employs a trehalose variant dubbed "quencher-trehalosefluorophore" (QTF, Fig.…”

The role of chemoenzymatic synthesis in advancing trehalose analogues as tools for combatting bacterial pathogens

“…73,96,97 Such detectable trehalose-based imaging probes have proven valuable for answering fundamental questions about mycobacterial cell envelope structure and metabolism, 60, 65, 66, 98-107 and are currently being pursued as tools for anti-mycobacterial drug development 47,108 and imaging of mycobacterial infections. 94,[109][110][111] Although progress on trehalose-based imaging probes over the last decade has been swift and exciting, a remaining impediment to the creation and adoption of these tools has been limited access to existing probes and the development of new probes. Chemoenzymatic synthesis has played an important role in addressing these problems, an illustrative example of which relates to the earliest trehalose analogues reported for imaging mycobacteria, FITC-Tre and the "clickable" TreAz series of analogues (structures shown above in Figure 4).…”

“…Some fluorogenic trehalose probes, synthesized via 6-TreAz as an intermediate, have even been shown to rapidly detect M. tuberculosis in patient sputum samples, demonstrating potential as point-of-care diagnostic tools. 109,110 However, these probes rely on optical imaging and thus are not suitable for specific in vivo imaging of mycobacteria in patients. Such a capability is not yet available but could transform how M. tuberculosis infections are managed in the clinic, including challenging cases involving recurrence, drug resistance, or disseminated disease.…”

Chemiluminescent Protease Probe for Rapid, Sensitive, and Inexpensive Detection of Live Mycobacterium tuberculosis

“… 12 , 13 New fluorescent probes have been developed to report on the activity of mycobacterial enzymes. For example, probes have been developed that target mycobacterial esterases, 14 sulfatases, 15 and trehalose mycolyltransesterases, 16 , 17 but these probes are nonspecific since they also label nontuberculous mycobacteria (NTMs) that may also be present in sputum. Probes activated by the Mtb β-lactamase BlaC have been shown to specifically label Mtb in patient sputum samples.…”