Identification of a Novel Mycobacterial Gene Involved in the Synthesis of a Phenolic Glycolipid and its Role in the Prevention of Phagosome Maturation

Abstract

Pathogenic Mycobacteria persist in an early endosome-like compartment by interfering with late endosomal fusion mediating factors. Studies have unraveled some of the mechanisms employed by mycobacteria to create a niche for themselves in macrophages, but it is widely accepted that they possess an arsenal of weapons to impede phagosome genesis.<br /> M. marinum has gained importance in recent years, as a model organism to study mycobacterial pathogenesis due to its phylogenetic closeness to M. tuberculosis. The infection it causes in its natural hosts display characteristic features of tuberculosis, exhibiting blocking of phagosome maturation and granuloma formation. <br /> To gain insight into the genes required for the inhibition of phagosome maturation, M. marinum transposon mutant library representing knock outs covering the entire genome was sifted for mutants defective in inhibiting phagosome maturation by designing an elegant screen, which employs magnetic separation. In this process we identified a number of mutants unable to inhibit phagosome maturation and characterised in detail one of these mutants (mutant P1). The colony morphology and sequence analysis revealed that the interrupted gene of mutant P1 (pmiA) is likely to be involved in lipid metabolism. The mutant also had a reduced intracellular survival as inferred from the in vitro bacterial survival experiments in HMDM and using mice as an in vivo model. The mutant completely reverted to its wild-type phenotype when complemented with the respective gene from wild-type M. marinum. Thin layer chromatography on the lipids isolated from the mutant showed that the disruption of the gene pmiA in mutant P1 leads to the loss of a glycolipid of the outer envelope of M. marinum (Robinson N et al., Infect Immun. 2007 Feb;75(2):581-91).<br /> The missing glycolipid was further characterised to be a phenolic glycolicpid (PGL) using mass spectrometry and nuclear magnetic resonance spectroscopy. In order to prove that the lipid is capable of inhibiting phagosome maturation, it was extracted from wild-type M. marinum, coated on to hydrophobic beads and chased into human monocyte derived macrophages (HMDM). Characterising the phagosomes containing the beads by western blot analysis and immunofluorescence microscopy proved the lipid to be a key molecule employed by virulent mycobacteria to inhibit phagosome maturation. <br /> Phagosomes were characterised employing an efficient adenoviral transfection system harbouring Rab-GFP fusion proteins to transfect primary phagocytes. This transfection system enables phagosome maturation to be studied efficiently by fluorescence microscopy in live cells, in contrast to immunostaining which can be performed only on fixed cells. <br /> The gene pmiA involved in the biosynthesis of the phenolic glycolipid shows little homology with the gene sequences available through genome databases. It also does not display any signature sequences of proteins with known functions. Therefore, an attempt was made to study its interacting proteins by using Histidine-tag pull down assay. Proteins interacting with pmiA were analyzed by mass spectrometry. A methyl transferase and an isocitrate lyase, both enzymes critically involved in lipid biosynthesis were found to interact with pmiA. Our results prove that genes involved in the synthesis of this phenolic glycolipid are ideal pharmacological targets to design drug interventions against tuberculosis.