Cholesteroid nature of free mycolic acids from M. tuberculosis.

Mycolic acids (MAs) are a major component of the cell walls of Mycobacterium tuberculosis and related organisms. These alpha-alkyl beta-hydroxy long fatty acids have been the subject of numerous studies for their immunological properties. We previously reported that an interaction between cholesterol and mycolic acids could be responsible for the low accuracy in the serodiagnosis of TB when using free mycolic acid in an ELISA assay. The aim of this work was to investigate if this interaction could be due to a similarity in the structural properties between mycolic acids and cholesterol. The investigation revealed that patient sera cross-reacted with mycolic acids and cholesterol in an ELISA experiment suggesting that both molecules may present related functionality in a similar structural orientation. This relation was further supported by the interaction of mycolic acids with Amphotericin B (AmB), a known binding agent to ergosterol and cholesterol. Using a resonant mirror biosensor, we observed that AmB recognised both cholesterol and mycolic acids. In addition, a specific attraction was observed between mycolic acid and cholesterol by the accumulation of cholesterol from liposomes in suspension onto immobilized mycolic acids containing liposomes, detected with a biosensor technique. Combined, these results suggest that mycolic acids can assume a three-dimensional conformation similar to a sterol. This requires that mycolic acid exposes its hydroxyl group and assumes rigidity in its chain structure to generate a hydrophobic surface topology matching that of cholesterol. A particular folded conformation would be required for this, of which a few different types have already been proven to exist in monolayers of mycolic acids.

A novel application of affinity biosensor technology to detect antibodies to mycolic acid in tuberculosis patients.

Tuberculosis has re-emerged as a global health problem due to co-infection with HIV and the emergence of drug-resistant strains of Mycobacterium tuberculosis. HIV co-infection introduced a 30% underestimation in TB diagnosis based on sputum analysis, calling for a reliable and fast serodiagnostic assay to assist in the management of TB in HIV-burdened populations. Serodiagnosis with mycobacterial lipid cell wall antigens gave promising results, in particular with LAM and cord factor. Free mycolic acids have also been considered because they are unique in structure to each species of Mycobacterium and can be economically extracted and purified. In a standard immunoassay such as ELISA, however, an unacceptable number of false positive and false negative test results were obtained. Here we report a much improved biosensor method to detect antibodies to mycolic acids in patient serum as surrogate markers of active tuberculosis. Mycolic acid (MA) liposomes were immobilized on a non-derivatized twin-celled biosensor cuvette and blocked with saponin. A high dilution of serum was used to calibrate the binding signal of the two cells, followed by contact with patient serum at a lesser dilution, but pre-incubated with either antigen-carrying, or empty liposomes. The serum, or the protein A purified IgG thereof, from sputum-positive tuberculosis patients could be inhibited from binding to the MA in the biosensor by prior incubation with MA-containing liposomes. The accuracy of the inhibition test was 84% if HIV-positive patients for whom a negative TB sputum analyses could not be relied upon to serve as a reference standard were excluded. If biosensor technology could be made suitable for high throughput screening, then it may provide the solution to the serodiagnosis of tuberculosis against a background of HIV.