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Molecular basis of phosphatidyl-myo-inositol mannoside biosynthesis and regulation in mycobacteria.

ABSTRACT: Phosphatidyl-myo-inositol mannosides (PIMs) are unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidyl-myo-inositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the structural basis of the lipoglycans (lipomannan and lipoarabinomannan), all important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy. Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis and regulation is still incomplete. Recent advances in the identification of key proteins involved in PIM biogenesis and the determination of the three-dimensional structures of the essential phosphatidyl-myo-inositol mannosyltransferase PimA and the lipoprotein LpqW have led to important insights into the molecular basis of this pathway.

SUBMITTER: Guerin ME

PROVIDER: S-EPMC2962455 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Molecular basis of phosphatidyl-myo-inositol mannoside biosynthesis and regulation in mycobacteria.

Guerin Marcelo E ME Korduláková Jana J Alzari Pedro M PM Brennan Patrick J PJ Jackson Mary M

The Journal of biological chemistry 20100827 44

Phosphatidyl-myo-inositol mannosides (PIMs) are unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidyl-myo-inositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the structural basis of the lipoglycans (lipomannan and lipoarabinomannan), all important molecules impli ...[more]

PMID: 20801880

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Project description:1. Addition of the bivalent ionophore A23187 to synaptosomes isolated from guinea-pig brain cortex and labelled with [(32)P]phosphate in vitro or in vivo caused a marked loss of radioactivity from phosphatidyl-myo-inositol 4-phosphate (diphosphoinositide) and phosphatidyl-myo-inositol 4,5-bisphosphate (triphosphoinositide) and stimulated labelling of phosphatidate. No change occurred in the labelling of other phospholipids. 2. In conditions that minimized changes in internal Mg(2+) concentrations, the effect of ionophore A23187 on labelling of synaptosomal di- and tri-phosphoinositide was dependent on Ca(2+) and was apparent at Ca(2+) concentrations in the medium as low as 10(-5)m. 3. An increase in internal Mg(2+) concentration stimulated incorporation of [(32)P]phosphate into di- and tri-phosphoinositide, whereas lowering internal Mg(2+) decreased labelling. 4. Increased labelling of phosphatidate was independent of medium Mg(2+) concentration and apparently only partly dependent on medium Ca(2+) concentration. 5. The loss of label from di- and tri-phosphoinositide caused by ionophore A23187 was accompanied by losses in the amounts of both lipids. 6. Addition of excess of EGTA to synaptosomes treated with ionophore A23187 in the presence of Ca(2+) caused a rapid resynthesis of di- and tri-phosphoinositide and a further stimulation of phosphatidate labelling. 7. Addition of ionophore A23187 to synaptosomes labelled in vivo with [(3)H]inositol caused a significant loss of label from di- and tri-phosphoinositide, but not from phosphatidylinositol. There was a considerable rise in labelling of inositol diphosphate, a small increase in that of inositol phosphate, but no significant production of inositol triphosphate. 8. (32)P-labelled di- and tri-phosphoinositides appeared to be located in the synaptosomal plasma membrane. 9. The results indicate that increased Ca(2+) influx into synaptosomes markedly activates triphosphoinositide phosphatase and diphosphoinositide phosphodiesterase, but has little or no effect on phosphatidylinositol phosphodiesterase.

Project description:Mycobacterium tuberculosis comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl-myo-inositol mannosides (PIMs) are glycolipids considered to be not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane-associated acyltransferase that transfers a palmitoyl moiety from palmitoyl coenzyme A (palmitoyl-CoA) to the 6-position of the mannose ring linked to the 2-position of inositol in PIM1/PIM2 We validate that the function of PatA is vital for M. tuberculosisin vitro and in vivo We constructed a patA conditional mutant and showed that silencing patA is bactericidal in batch cultures. This phenotype was associated with significantly reduced levels of Ac1PIM2, an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the patA gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for M. tuberculosis growth in vitro and in vivo Altogether, the experimental data highlight the significance of the early steps of the PIM biosynthetic pathway for M. tuberculosis physiology and reveal that PatA is a novel target for drug discovery programs against this major human pathogen.IMPORTANCE Tuberculosis (TB) is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of M. tuberculosis, the etiologic agent of TB, emphasizes the need to identify new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined that PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in M. tuberculosis These results highlight the importance of early steps of the PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target.

Dataset Information

Molecular basis of phosphatidyl-myo-inositol mannoside biosynthesis and regulation in mycobacteria.

Publications

Molecular basis of phosphatidyl-myo-inositol mannoside biosynthesis and regulation in mycobacteria.

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