Structure–function relationships of the antigenicity of mycolic acids in tuberculosis patients

Abstract

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids

“…Although mycolic acids are ideal conservative compounds for Mtb confirmation studies, some territory, culturing, time of growth or physiological state related differences in their compositions were observed. Experimental data demonstrated that mycolic acids from Mtb, consisting mainly of alpha-, keto-, and methoxy-mycolates, contain mixtures of homologs with different chain lengths or stereochemistries around the functional groups in the main chain (Beukes et al 2010). These variables can possibly cause slight differences in the content of the single mycolic acid.…”

Towards understanding the functional diversity of cell wall mycolic acids of Mycobacterium tuberculosis

“…Antigenicity was also found to depend critically on the structure of the mycolic motif. In particular, methyl esterification of the carboxylic acid in the motif destroyed antigenicity altogether [126], possibly indicating a critical role of the carboxylic acid in stabilizing an antigenic conformational fold of the MA by hydrogen bonding with the oxygenated distal functional group.…”

“…8 is that none of the MA isomers examined showed any significant difference in distinguishing between sera from TB-positive and TB-negative patients. The same applied to isomers of the keto-and alpha-MA classes [126].…”

“…Such monospecific scFv specificities were also selected from the chicken antibody gene library by Beukes et al [126], showing that the recognition of MA by antibody does not necessarily manifest as cross-reactivity to cholesterol. Likewise, scFv antibody fragments to cholesterol did not necessarily cross-react with MA.…”

Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis

“…The nanospray voltage was Ϫ 1.35kV (negative ionization) All four antibodies were subsequently tested against a panel of synthetic and purifi ed lipids. Earlier studies had indicated that mycolic acid might have a similar 3D structure to cholesterol and evidence of cross-reactivity to cholesterol was observed in anti-mycolic acid antibodies previously isolated from a chicken phage display library ( 22,23 ). We therefore tested all four antibodies against purifi ed cholesterol and oxidized cholesterol derivatives.…”