title: Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan.
abstract: Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-(14)C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM.
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, 1081 BT Amsterdam, The Netherlands.
authors.forename: Nicole N
authors.initials: NN
authors.lastname: Driessen
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, 1081 BT Amsterdam, The Netherlands.
authors.forename: Esther J M
authors.initials: EJM
authors.lastname: Stoop
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, 1081 BT Amsterdam, The Netherlands.
authors.forename: Roy
authors.initials: R
authors.lastname: Ummels
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston B15 2TT, UK.
authors.forename: Sudagur S
authors.initials: SS
authors.lastname: Gurcha
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston B15 2TT, UK.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Mishra
authors.affiliation: Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France.|CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 Route de Narbonne, F-31077 Toulouse, France.
authors.forename: Gérald
authors.initials: G
authors.lastname: Larrouy-Maumus
authors.affiliation: Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France.|CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 Route de Narbonne, F-31077 Toulouse, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Nigou
authors.affiliation: Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France.|CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 Route de Narbonne, F-31077 Toulouse, France.
authors.forename: Martine
authors.initials: M
authors.lastname: Gilleron
authors.affiliation: Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France.|CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 Route de Narbonne, F-31077 Toulouse, France.
authors.forename: Germain
authors.initials: G
authors.lastname: Puzo
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, 1081 BT Amsterdam, The Netherlands.
authors.forename: Janneke J
authors.initials: JJ
authors.lastname: Maaskant
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, 1081 BT Amsterdam, The Netherlands.
authors.forename: Marion
authors.initials: M
authors.lastname: Sparrius
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston B15 2TT, UK.
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, 1081 BT Amsterdam, The Netherlands.
authors.forename: Wilbert
authors.initials: W
authors.lastname: Bitter
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, 1081 BT Amsterdam, The Netherlands.
authors.forename: Christina M J E
authors.initials: CMJE
authors.lastname: Vandenbroucke-Grauls
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, 1081 BT Amsterdam, The Netherlands.
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Appelmelk
mesh_terms: D000215:Acylation; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D008358:Mannose / Q000096:biosynthesis* / Q000737:chemistry; D008364:Mannosyltransferases / Q000378:metabolism; D009154:Mutation; D019910:Mycobacterium marinum / Q000201:enzymology* / Q000235:genetics
chemical_list: D008070:Lipopolysaccharides; C050016:lipoarabinomannan; D000217:Acyltransferases; D008364:Mannosyltransferases; D008358:Mannose