PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria.
Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.
Morita
Yasu S
YS
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
Sena
Chubert B C
CB
Waller
Ross F
RF
Kurokawa
Ken
K
Sernee
M Fleur
MF
Nakatani
Fumiki
F
Haites
Ruth E
RE
Billman-Jacobe
Helen
H
McConville
Malcolm J
MJ
Maeda
Yusuke
Y
Kinoshita
Taroh
T
D000595:Amino Acid Sequence; D049109:Cell Proliferation; D002473:Cell Wall / Q000378:metabolism; D002474:Cell-Free System; D016680:Genome, Bacterial; D006801:Humans; D008358:Mannose / Q000737:chemistry; D008362:Mannosides / Q000737:chemistry*; D008364:Mannosyltransferases / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000378:metabolism; D010710:Phosphates; D010716:Phosphatidylinositols / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D008362:Mannosides; D010710:Phosphates; D010716:Phosphatidylinositols; D008364:Mannosyltransferases; D008358:Mannose