CD1b-mediated T cell recognition of a glycolipid antigen generated from mycobacterial lipid and host carbohydrate during infection.

T cells recognize microbial glycolipids presented by CD1 proteins, but there is no information regarding the generation of natural glycolipid antigens within infected tissues. Therefore, we determined the molecular basis of CD1b-restricted T cell recognition of mycobacterial glycosylated mycolates, including those produced during tissue infection in vivo. Transfection of the T cell receptor (TCR) alpha and beta chains from a glucose monomycolate (GMM)-specific T cell line reconstituted GMM recognition in TCR-deficient T lymphoblastoma cells. This TCR-mediated response was highly specific for natural mycobacterial glucose-6-O-(2R, 3R) monomycolate, including the precise structure of the glucose moiety, the stereochemistry of the mycolate lipid, and the linkage between the carbohydrate and the lipid. Mycobacterial production of antigenic GMM absolutely required a nonmycobacterial source of glucose that could be supplied by adding glucose to media at concentrations found in mammalian tissues or by infecting tissue in vivo. These results indicate that mycobacteria synthesized antigenic GMM by coupling mycobacterial mycolates to host-derived glucose. Specific T cell recognition of an epitope formed by interaction of host and pathogen biosynthetic pathways provides a mechanism for immune response to those pathogenic mycobacteria that have productively infected tissues, as distinguished from ubiquitous, but innocuous, environmental mycobacteria.

Structural requirements for glycolipid antigen recognition by CD1b-restricted T cells.

The human CD1b protein presents lipid antigens to T cells, but the molecular mechanism is unknown. Identification of mycobacterial glucose monomycolate (GMM) as a CD1b-presented glycolipid allowed determination of the structural requirements for its recognition by T cells. Presentation of GMM to CD1b-restricted T cells was not affected by substantial variations in its lipid tails, but was extremely sensitive to chemical alterations in its carbohydrate or other polar substituents. These findings support the view that the recently demonstrated hydrophobic CD1 groove binds the acyl chains of lipid antigens relatively nonspecifically, thereby positioning the hydrophilic components for highly specific interactions with T cell antigen receptors.

Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis.

Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.