Structural and serological characterization of the major glycolipid from Rothia mucilaginosa Mariola Pas ´ciak a , Otto Holst b , Buko Lindner c , Magdalena Mierzchaya a , Anna Grzegorzewicz a , Halina Mordarska a , Andrzej Gamian a, * a Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, PL-53-114 Wrocy aw, Poland b Division of Structural Biochemistry, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 4a, 23845 Borstel, Germany c Division of Biophysics, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 10, 23845 Borstel, Germany Received 5 May 2004; received in revised form 6 August 2004; accepted 20 August 2004 Available online 9 September 2004 Abstract Structural studies on the major glycolipid isolated from Rothia mucilaginosa were carried out utilising specific chemical degradation, NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI TOF-MS). The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside a-d-Manp -(1Y3)-a-d- Manp -(1Y3)-sn -Gro (Man A-Man B-Gro), of which Man B was esterified at O-6 by a fatty acid residue. A second fatty acid substituted the secondary methylene position of the glycerol residue, in contrast to the glycolipid previously found in R. dentocariosa and Saccharopolyspora strains, in which the second fatty acid esterified the primary methylene position of glycerol. Results of the ELISA experiment with rabbit specific antibacterial sera indicate that these two major glycolipids are antigenic, and the patterns of serological reactivity are similar but not identical. D 2004 Elsevier B.V. All rights reserved. Keywords: Rothia mucilaginosa; Stomatococcus ; Opportunistic agent; Glycolipid; Structure; NMR; Dimannosylacylmonoglyceride 1. Introduction Rothia mucilaginosa , previously known as Stomatococ- cus mucilaginosus , is a pathogen responsible for a variety of cases of opportunistic infections [1–7]. Immunocompro- mised patients are very often infected with this pathogen, leading to endocarditis [1,5,8–11], sepsis [12,13], peritonitis [5] and infections associated to tissue and organ trans- plantations and implants. Also, complications are caused in patients with tumors, diabetes, and other diseases [14]. The microorganism was first isolated from milk by Migula in 1900 and called Micrococcus mucilaginosus [15]. Later, it was called Staphylococcus salivarius for a short time, then again M. mucilaginosus and in 1982 new chemotaxonomic and genotaxonomic methods allowed to classify the strain into a separate genus Stomatococcus with its only species S. mucilaginosus [16]. Recently, S. mucilaginosus was again integrated into a new genus, Rothia , and named R. mucilaginosa [17]. The classification of this bacterium was difficult due to the similarity to micrococci, staphylococci and other Gram-positive cocci like streptococci or enterococci. The natural environment of R. mucilaginosa is the oral cavity and the upper respiratory tract, but it was also isolated from throat, lungs, blood [1,8,18,19] and dental plaques [20]. The microorganism is a nonsporulating, nonmotile 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.08.004 Abbreviations: BHI, brain heart infusion; FP, furazolidone-peptone agar; FA, fatty acid; Gro, glycerol; HMQC, 1 H-detected heteronuclear multiple quantum correlation spectroscopy; DQF-COSY, double quantum filtered correlated spectroscopy; DPG, diphosphatidylglycerol; PG, phos- phatidylglycerol; DGDG, diglucosyldiglyceride; DMDG, dimannosyldigly- ceride; DMAMG, dimannosylacylmonoglyceride; PI, phosphatidylinositol; TAG, tetraacylglucose; T, type strain * Corresponding author. Tel.: +48 71 3371172; fax: +48 71 3371382. E-mail address: gamian@immuno.iitd.pan.wroc.pl (A. Gamian). Biochimica et Biophysica Acta 1675 (2004) 54 – 61 http://www.elsevier.com/locate/bba