One in three people on earth are living with latent Mycobacterium tuberculosis (Mtb) infection. Every year 8 million more people develop active tuberculosis (TB) and 2 million die from the disease. Iron is an essential nutrient for the growth and pathogenesis of Mtb and is required for the function of many important enzymes and metabolic processes. Mtb uses iron-chelating molecules called siderophores to remove iron from the host. Although siderophores are required for Mtb virulence1, they have never before been detected in vivo and their precise role and level of expression in TB disease remains elusive. An electrochemical biosensor is proposed for the detection of these siderophores to elucidate their role and quantitatively test for their presence in diseased tissue and clinical samples. This technology could ultimately lead to new diagnostic tools for detection of Mtb infection.
Mtb produces two principle types of siderophores: the lipophilic ‘cell-associated’ mycobactins2, and the relatively water-soluble carboxymycobactins3,4. The variability of the long fatty acid component of their structures makes them a challenging detection target. Lipophilic mycobactins were extracted from Mtb culture supernatants and cell pellets and analyzed by HPLC-MS to identify the primary species of mycobactins produced. Supernatant extracts yielded the greatest quantities of mycobactins. Lipophilic mycobactins have previously been shown to diffuse freely in and around macrophages5, suggesting that they may be transported out of TB-infected cells and could be detected in tissues of infected individuals.
Lipocalin 2 (Lcn2) is a human neutrophil protein that has been shown to bind the carboxymycobactins of Mtb6 and has been chosen as the receptor for mycobactin capture as part of the biosensor. For this project, Lcn2 was cloned from the human cDNA and expressed in BL-21 cells with a hexahistidine tag on the N-terminus. This tag will be used to anchor Lcn2 to the electrode surface.
Separate experiments indicated that the specific reduction-oxidation activity of ferric iron bound to mycobactin can be successfully interrogated using cyclic voltammetry. This was performed on a glassy carbon electrode in a 1:1 ethanol:PBS solution containing 100 mM potassium chloride and 1 mM ferric mycobactin J (MJ, Allied Monitor).
This evidence supports the feasibility of a biosensor for rapid detection of Mtb siderophores. Testing of clinical samples could lead toward applications of this sensor as a low-cost diagnostic tool for TB diagnosis in resource-poor settings.
1. J. J. De Voss, at al., PNAS, 2000, 97, 1252-7.
2. G. A. Snow, Biochem J, 1965, 97, 166-175.
3. S. J. Lane, et al., Tetrahedron Letters, 1995, 36, 4129-32.
4. J. Gobin, et al., PNAS, 1995, 92, 5189-93.
5. M. Luo, et al., Nat Chem Biol, 2005, 1, 149-53.
6. M. A. Holmes, et al., Structure, 2005, 13, 29-41.