March 2025 – Nasim Najjarzadeh (PhD), Marketing Manager

Trehalose

Trehalose is a non-reducing disaccharide consisting of two glucose residues connected by a 1,1-α,α-glycosidic linkage (Fig. 1). It is abundant in nature, found in bacteria, yeast, fungi, plants, and invertebrates (1). The distinctive structure and exceptional stability of trehalose contribute to its main biological function of stress protection (1). Trehalose stabilizes a wide range of biomaterials, including proteins, DNA, cells, and tissues, making it valuable in pharmaceutical, food, and cosmetic industries (1).

Fig 1) Trehalose

Application of Trehalose in Diagnostics and Medicine

Trehalose is crucial for the growth and virulence of significant pathogens like Mycobacterium tuberculosis. Due to its importance in various pathogens and insect vectors, and its absence in mammals, recent research has focused on targeting trehalose metabolism with analogues for disease diagnosis, treatment, or prevention of transmission. However, the synthesis of trehalose analogues is challenging due to the unique 1,1-α,α-glycosidic linkage of trehalose, limiting their availability for research and commercial applications. Although mammals lack trehalose biosynthesis pathways, they possess trehalase, which hydrolyzes ingested trehalose to glucose, impacting the applications of trehalose and its analogues. In addition to its biosynthetic and degradative pathways, trehalose is involved in other pathways in some organisms. Notably, it plays a crucial role in constructing the complex cell envelope of Corynebacterineae, which includes mycobacterial pathogens like Mycobacterium tuberculosis (1).

Role of Trehalose in Mycobacterium Tuberculosis

Trehalose is vital for the growth and virulence of Mycobacterium tuberculosis, a globally significant pathogen. It functions not only as an energy storage molecule and stress protectant but also plays a crucial role in constructing the mycobacterial cell envelope. Trehalose is anchored into the mycobacterial cell wall as mono-(TMM) or di-(TDM) mycolates by the action of the extracellular proteins Ag85A, Ag85B, and Ag85C. These proteins are the most abundantly secreted M. tuberculosis proteins in vitro, accounting for up to 41% of the total protein in culture supernatant and individual knockouts of the genes coding for single members of the Ag85 family significantly affect total cellular mycolic acid content. Mycolic acids are synthesized in the cytoplasm and esterified to the disaccharide trehalose to generate TMM. After crossing the plasma membrane, TMM donates its mycoloyl group to either the terminal residues of the arabinogalactan polymer, generating arabinogalactan-linked mycolates (AGM), or to another TMM molecule, generating trehalose dimycolate (TDM). Ag85 isoforms catalyze the reversible transesterification reaction between two units of TMM (generating TDM and free trehalose), and can also introduce mycolates into arabinogalactan to form the base polymer of the cell wall.

TMM and TDM are significant for M. tuberculosis, capable of inducing granuloma formation even in the absence of infection and mycolic acids are essential for the bacterial outer membrane structure, virulence, and persistence within the host (1, 2).

Fig 2)  Ag85A, Ag85B and Ag85C catalyze transesterification of trehalose, TDM and TMM.

Trehalose Analogues

Given the importance of trehalose in various pathogens and its absence in mammals, trehalose analogues have garnered interest for diagnostic, therapeutic, and biopreservation applications. Trehalose analogues containing detectable moieties, such as 14C-labeled, stable isotope-labelled, fluorescent-labeled, azide-labeled, alkyne-labeled, and 18F-labeled trehalose, are valuable tools for studying trehalose metabolism and detecting pathogens. Among these, FITC-Trehalose, a fluorescent trehalose probe, has demonstrated efficacy in labeling live M. tuberculosis cells through incorporation into surface glycolipids. This capability allows for the specific imaging of intracellular M. tuberculosis within macrophages, highlighting the potential of fluorescent trehalose probes in tuberculosis diagnostics.

Fluorescent-labelled trehalose

In 2011, FITC-Tre, was introduced as the first trehalose-based fluorescent probe (2).

Fig 3) (A) Structure of FITC-Tre (26). (B) Metabolic labeling of M. tuberculosis within host macrophages using FITC-Tre. Scale bar, 5μm

FITC-Tre is a ketoside trehalose analogue with a thiourea-linked fluorescein group at its 2’-position, capable of reporting on trehalose metabolism in living cells through fluorescence microscopy. The initial study demonstrated FITC-Tre’s ability to label live M. tuberculosis cells via Ag85-mediated incorporation into surface glycolipids like TDM. This work revealed two key insights: extracellular Ag85 has high substrate tolerance, and FITC-Tre could be used to track mycobacterial trehalose metabolism in vivo because the Ag85 pathway is absent from humans and other mammals. FITC-Tre was successfully used for imaging intracellular M. tuberculosis within macrophages (fig 3-B), suggesting its potential for detecting and investigating M. tuberculosis in complex environments. This concept is now being advanced through the development of trehalose-based tuberculosis diagnostic tools. FITC-Tre marked a significant advancement in trehalose analogues for studying mycobacteria and has inspired next-generation fluorogenic trehalose analogues that produce fluorescence only upon binding to mycobacteria.

Challenges and Future Directions

Despite the promising applications of trehalose analogues, their synthesis remains challenging. The unique 1,1-α,α-glycosidic linkage of trehalose complicates analogue synthesis, limiting access to these compounds for research and commercial purposes. Advances in both chemical and chemoenzymatic synthesis methods are needed to overcome these challenges.

Future research should focus on scaling up the production of trehalose analogues and exploring the applications of detectable trehalose analogues in diagnostics. Additionally, a better understanding of the metabolism and specificity of trehalose-based probes in various microbial species and human contexts will be crucial for their successful implementation.

Conclusion

Trehalose analogues represent an exciting class of molecules with significant potential in biomedicine, biotechnology, and basic research. Continued advancements in their synthesis and application will be essential for harnessing their full potential in diagnosing and treating diseases, preserving biomaterials, and understanding trehalose metabolism across diverse organisms.

References

1) O’Neill MK, Piligian BF, Olson CD, Woodruff PJ, Swarts BM. Tailoring Trehalose for Biomedical and Biotechnological Applications. Pure Appl Chem. 2017 Sep;89(9):1223-1249. doi: 10.1515/pac-2016-1025. Epub 2017 Jan 11. PMID: 29225379; PMCID: PMC5718624.

2)  Backus KM, Boshoff HI, Barry CS, Boutureira O, Patel MK, D’Hooge F, Lee SS, Via LE, Tahlan K, Barry CE 3rd, Davis BG. Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis. Nat Chem Biol. 2011 Apr;7(4):228-35. doi: 10.1038/nchembio.539. Epub 2011 Mar 6. PMID: 21378984; PMCID: PMC3157484.