A processive lipid glycosyltransferase in the small human pathogen Mycoplasma pneumoniae: involvement in host immune response

Abstract

SummaryThe human pathogen Mycoplasma pneumoniae has a very small genome but with many yet not identified gene functions, e.g. for membrane lipid biosynthesis. Extensive radioactive labelling in vivo and enzyme assays in vitro revealed a substantial capacity for membrane glycolipid biosynthesis, yielding three glycolipids, five phosphoglycolipids, in addition to six phospholipids. Most glycolipids were synthesized in a cell protein/lipid-detergent extract in vitro; galactose was incorporated into all species, whereas glucose only into a few. One (MPN483) of the three predicted glycosyltransferases (GTs; all essential) was both processive and promiscuous, synthesizing most of the identified glycolipids. These enzymes are of a GT-A fold, similar to an established structure, and belong to CAZy GT-family 2. The cloned MPN483 could use both diacylglycerol (DAG) and human ceramide acceptor substrates, and in particular UDPgalactose but also UDP-glucose as donors, making mono-, di-and trihexose variants. MPN483 output and processitivity was strongly influenced by the local lipid environment of anionic lipids. The structure of a major b1,6GlcbGalDAG species was determined by NMR spectroscopy. This, as well as other purified M. pneumoniae glycolipid species, is important antigens in early infections, as revealed from ELISA screens with patient IgM sera, highlighting new aspects of glycolipid function.

Expression and Characterization of a Mycoplasma genitalium Glycosyltransferase in Membrane Glycolipid Biosynthesis

“…With UDP-Gal as glycosyl donor, DAG and Gal␤DAG were good acceptors, the latter being preferred to give Gal␤1,6Gal␤DAG. With UDP-Glc as donor, DAG and Gal␤DAG were acceptors, but not Glc␤DAG (38). Moreover, the main glycolipid synthesized in vivo by recombinant E. coli cells expressing mpn483 was identified as Glc␤1,6Gal␤DAG.…”

“…GT MG517 is homologous to the M. pneumoniae enzyme encoded by mpn483 gene, reported to produce similar glycolipids (38). However, both enzymes show subtle differences in substrate specificity.…”

“…BLAST screening identified homologues in other Mycoplasma species, being those from M. pneumoniae the closest relatives. The M. genitalium mg517 gene product shares 77% similarity with the M. pneumoniae MPN483 protein, recently identified as a processive GT-synthesizing galactosyl and glucosyl-␤1,6-galactosyldiacylglycerols (38). The other two genes, mg025 and mg060 (orthologs of mpn028 and mpn075), have unknown function.…”

“…However, genome-sequenced mycoplasmas (more than 18 different species) contain mainly GT2 enzymes. Experimentally, only the MPN483 gene product from M. pneumoniae has been identified as a glycolipid synthase (38). It is responsible for the synthesis of most of the glycoglycerolipids found in the plasma membrane (processive enzyme with broad specificity), but this study characterized the enzyme in crude membrane extracts, where other membrane components may interfere or regulate the observed activity.…”

“…Two different GTs belonging to family GT4 have been identified as mono-and diglucosyldiacylglycerol synthases, respectively, responsible for the sequential glycosylation of the precursor diacylglycerol and regulation of the ratio between non-bilayer and bilayer-forming glycolipids (32)(33)(34)(35)(36)(37). More recently, Mycoplasma pneumoniae has been shown to contain a processive GT2 producing mainly galactoglycerolipids (38).…”