Microbial genome sequencing projects have revealed an unanticipated variety of metabolic and cellular capabilities. Therefore, it is not surprising that microbes can successfully adapt to different habitats. The key to the success of Mycobacterium tuberculosis (Mtb) as a pathogen has been its ability to reside and proliferate inside host macrophages despite their antimicrobial properties. Pathogenic mycobacteria are endowed with a cell envelope of remarkable molecular architecture. Along with complex lipids and polysaccharides, the mycobacterial cell envelope is associated with numerous proteins that influence cellular structure and the pathogen's interactions with host cells. Given that the cell envelope constitutes the key interface between pathogen and host, cell wall–associated proteins and lipids are presumed to be the determinants of pathogenesis and immunogenicity. There is growing evidence to suggest that differential expression of molecules on the cell surface could lead to phenotypic heterogeneity, resulting in altered virulence patterns. Our group is interested in examining the molecular mechanisms by which mycobacteria modulate expression of their cell wall components—both lipids and proteins—in response to changes in the host environment.
The mycobacterial genome has a large repertoire of lipid-modifying enzymes. The genome sequence of Mtb revealed an array of proteins homologous to polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These are large, multifunctional enzymes that assemble simple building blocks to produce complex secondary metabolites. Mostly PKSs and NRPSs from Streptomyces and fungi have been studied; in these organisms the enzymes biosynthesize pharmaceutically important metabolites. Mycobacteria, on the other hand, are not known to produce such secondary metabolites, but they are known for the large collection of lipids that constitute their unusual cell envelope. Our studies have provided insights into the mechanism by which mycobacteria use these proteins to produce complex lipids. By combining genetic, biochemical, and computational approaches, we have been able to reconstruct metabolic pathways, thus providing the precise roles of proteins involved in lipid biosynthesis. To rationalize and predict metabolites biosynthesized by multifunctional PKS and NRPS proteins, we have developed Web-based software packages in collaboration with Debasisa Mohanty of the Bioinformatics Center, National Institute of Immunology, New Delhi (http://www.nii.res.in/nrps-pks.html). This software facilitates correct identification of catalytic domains and predicts their substrate specificity.
PKS and NRPS proteins are characteristically organized as large genetic clusters, which include several accessory protein domains—tailoring enzymes that modify the core structure assembled by PKS and NRPS proteins. Modifications include oxidation, reduction, glycosylation, acylation, and alkylation and are essential for the lipids' bioactivity. Mycobacterial genomes encode a number of such modifying enzymes; our current interest is to understand the functional interplay that dictates biosynthesis of the final metabolic product. In a recent study, we identified a new family of fatty acyl-AMP ligases (FAALs), which activate fatty acids to their corresponding acyl-adenylates and transfer them directly to the PKS proteins. We speculate that these functional modifications that lead to a subtle cell wall–remodeling process may be crucial for sustained Mtb infections.
Another interesting aspect of mycobacterial lipids is their ability to elicit complex inflammatory immune responses. It is tempting to speculate that mycobacteria may establish their niche in the host cell by varying the phenotypic expression of these lipid metabolites. The expressions could be precisely calibrated to achieve a particular equilibrium, which would result in a chronic state that is permissive for both bacilli and the host. Our recent studies suggest remarkable promiscuity for some of these modifying enzymes in terms of their substrate specificity. Clearly, the metabolic fluxes in vivo could dictate the biosynthesis of metabolic products.
Lipid-biosynthetic enzymes could prove to be a promising target for developing antitubercular drugs. Polyketide enzymes are excellent targets for drug discovery for many reasons. However, no specific inhibitors of PKSs are known. FAAL proteins that feed long-chain fatty acids to PKS enzymes for the production of complex lipids could also prove to be an important target. The "one disease–one drug–one target" paradigm that has dominated thinking in the pharmaceutical industry for the past few decades is now being increasingly challenged by the discovery of compounds that bind to more than one target. It is now widely acknowledged that high specificity for a single target might not always deliver the required efficacy for a given side effect profile. An interesting proposition is to combine two selective ligands. Another possibility is to design a single chemical entity that could simultaneously target a similar family of enzymes. We are interested in developing such multiple-ligand inhibitors.
The identification and characterization of molecular mechanisms that generate functional heterogeneity can significantly expand our understanding of how pathogens evolve their metabolic pathways to generate molecular diversity and how they use this diversity to respond to the complex challenges presented by the host immunity.
This work is also partly supported by the Department of Biotechnology, India.
Last updated April 2007
