Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Jul 1.
Published in final edited form as: Biochim Biophys Acta. 2013 Apr 2;1831(7):1239–1249. doi: 10.1016/j.bbalip.2013.03.012

Structural Identities of Four Glycosylated Lipids in the Oral Bacterium Streptococcus mutans UA159

Larry Sallans a, José-Luis Giner b, David J Kiemle b, Jenny E Custer c, Edna S Kaneshiro c,*
PMCID: PMC3672342  NIHMSID: NIHMS463387  PMID: 23562838

Abstract

The cariogenic bacterium Streptococcus mutans is an important dental pathogen that forms biofilms on tooth surfaces, which provide a protective niche for the bacterium where it secretes organic acids leading to the demineralization of tooth enamel. Lipids, especially glycolipids are likely to be key components of these biofilm matrices. The UA159 strain of S. mutans was among the earliest microorganisms to have its genome sequenced. While the lipids of other S. mutans strains have been identified and characterized, lipid analyses of UA159 have been limited to a few studies on its fatty acids. Here we report the structures of the four major glycolipids from stationary-phase S. mutans UA159 cells grown in standing cultures. These were shown to be monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG), diglucosylmonoacylglycerol (DGMAG) and, glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). The structures were determined by high performance thin-layer chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. The glycolipids were identified by accurate, high resolution, and tandem mass spectrometry. The identities of the sugar units in the glycolipids were determined by a novel and highly efficient NMR method. All sugars were shown to have α-glycosidic linkages and DGMAG was shown to be acylated in the sn-1 position by NMR. This is the first observation of unsubstituted DGMAG in any organism and the first mass spectrometry data for GPDGDAG.

Keywords: Bacterium, glycolipids, lipidomics, dental pathogen

1. Introduction

The oral bacterium Streptococcus mutans synthesizes and releases a number of phospholipids and glycolipids [1,2] believed to play a role in the formation of dental plaque biofilms. S. mutans also produces organic acids within these biofilms covering teeth surfaces and causes demineralization of the tooth enamel that contributes to the formation of caries [3,4]. Pieringer and his colleagues [1,2] earlier identified the major lipids in the S. mutans BHT and FA-1 strains grown in a chemically defined medium, and reported that as much as 30% of the lipids synthesized by S. mutans were released from the cell and that the relative percent composition of the cellular and extracellular lipid classes were similar. They identified diacylglycerol (DAG), phosphatidylglycerol, (PG), diphosphatidylglycerol (cardiolipin, CL), aminoacyl-PG and three glycolipids. The glycolipids identified were monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG) and glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). That the bacteria synthesized these lipids was demonstrated by metabolic radiolabeling experiments in which [14C]glycerol was incorporated into each of these lipid components [1]. Since those reports, the genome of S. mutans strain UA159 was sequenced [3] and this strain is now being studied in detail with respect to gene expression, biochemistry, and physiology [4-11]. Since it is important to document the biochemical nature of this strain, in this report we focus on detailed characterizations of four glycolipids of the UA159 strain providing definitive structural identifications by high performance thin-layer chromatography (HPTLC), high resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Other detailed studies on the effects of culture age and pH on lipid class and fatty acid compositions are part of a separate report (Custer, J.E., Goddard, B.D., Kaneshiro, E.S., in preparation).

2. Materials and methods

2.1 Bacterial cultures and lipid extraction

Streptococcus mutans strain UA159 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown at 37 °C in 5% CO2 in Fernbach flasks containing 500 mL of 3.7 % brain heart infusion (BHI) medium (Becton, Dickinson and Company; Sparks, MD). Culture growth was monitored spectrophotometrically at Abs600 nm. After 8 h, cultures were in log phase and after 20 h they were in stationary phase. Most structural analyses in the present study were performed on stationary-phase cells (20-h cultures) when most lipid classes were detectable and sufficient amount of material could be isolated. Organisms were harvested by vigorous mixing to suspend cells attached to the bottom of flasks in biofilms. The suspended cells were pelleted by centrifugation at 5,000 × g for 15 min at 4 °C. After centrifugation, the supernatant was discarded and the cell pellet was washed twice with double-distilled water (ddH2O). Total lipids were extracted by adding chloroform and methanol to the packed cell pellet according to the protocol of Bligh and Dyer [12]. To test whether the BHI culture medium contain the lipids found in S. mutans, 18.5 g of BHI powder (equivalent to that in a 500-ml culture) was extracted for lipids with CHCl3:MeOH (2:1, v/v) and analyzed as a control.

All solvents for lipid extraction, fractionation and isolation, except those used for final MS and NMR analysis, contained the antioxidant butylated hydroxytoluene (BHT). After extraction for 2 h at room temperature, the suspension was centrifuged at 5,000 × g for 15 min then the supernatant containing total extracted lipids was removed and purified by biphasic partitioning [13]. The volume of the total lipid fraction was reduced (Brinkmann Rotavapor, BÜCHI, Flawil, Switzerland) and transferred to a 4-dram, screw-capped, Teflon-lined shell vial and dried under N2 (N-Evap, Organomation, Shrewsbury, MA).

2.2 Isolation of lipid classes

Total lipids were fractionated by adsorption column chromatography (Unisil, Clarkson Chemical, South Williamsport, PA) by first eluting with CHCl3 followed by elution of the polar lipid fraction with CH3OH. The components in the polar lipid fraction were isolated by 1- or 2- dimensional thin-layer chromatography (TLC) using high-performance HPTLC Silica gel 60, (EMD Chemicals, Darmstadt, Germany) or Al-backed Silica gel 60 (Alltech Associates Inc., Deerfield, IL) plates. The solvent system for development in the first dimension was CHCl3:CH3OH:ddH2O (65:25:4, v/v/v); CHCl3:acetone:CH3OH: acetic acid (HAc):ddH2O (50:20:10:10:5, v/v/v/v/v) was used for the second dimension. Initial identification of lipid spots was performed by 5% phosphomolybdate in ethanol and I2 vapor as a general lipid stains, orcinol for sugars, ninhydrin for amino and imino groups, the Dragendorf reagent for choline and a reagent described by Dittmer & Lester [14] for phosphorus. To isolate individual components, plates were wet with ddH2O to locate spots in 2-D HPTLC or bands in 1-D HPTLC and the lipid components were isolated by scraping, eluting with CHCl3:CH3OH (1:2, v/v) and dried under N2.

2.3 Mass spectrometry

Analyses of the isolated glycolipids were performed with a Thermo Scientific LTQ-FT™ using the static nanospray source and New Objective PicoTip™ emitters (2 mm tip). Nanospray samples were prepared by first dissolving the dried lipids in CHCl3 followed by the addition of MeOH for negative ion, or MeOH containing 0.5 mM NaI for positive ion giving a final ratio of CHCl3:MeOH of between 1:1 or 1:2 volume to volume. The glycolipids gave strong Na+ adducts under positive ion conditions. Negative ion mode was used for analyzing the phosphoglycolipid.

The source voltage was held at 1.70 kV with a capillary temperature of 175 °C. High mass accuracy measurements of both the nanosprayed ions and product ions arising from collision-induced dissociation was performed in the Fourier transform ion cyclotron resonance (FT-ICR) portion of the LTQ-FT™ with the resolution generally set at 100,000 (at m/z 400) although some situations required using 500,000 resolving power. Errors in mass accuracy were below 1 ppm with 235±129 ppb as the average of absolute errors. Collision-induced dissociation (MSn) was performed in the linear ion trap portion of the system. FT-ICR MSn mass errors were slightly higher due to the lower product ion intensities (484±255 ppb).

2.4 NMR spectroscopy

2.4.1 General

NMR spectra were measured using a 600 MHz Bruker Avance instrument at 30 °C. The chemical shifts in solvents containing CDCl3 were calibrated to residual CHCl3 (7.260 ppm); in solvents containing d6-DMSO, they were calibrated to residual d5-DMSO (2.500 ppm); in 2M D2SO4/D2O, DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid, 0.015 ppm) was used for calibration.

NMR procedures employed included COrrelation SpectroscopY (COSY), Nuclear Overhauser Effect SpectroscopY (NOESY), Rotating-Frame Nuclear Overhauser Effect SpectroscopY (ROESY) and TOtal Correlation Spectroscopy (TOCSY).

2.4.2 Sugar Analysis

A sample was digested with 2M D2SO4/D2O at 100 °C for 1 h. The resulting hydrolysate was analyzed directly by 1H-NMR. The spectrum, in particular the anomeric region, was compared to reference spectra of different sugars and was found to match that of glucose [15]. Under these conditions, glucose exhibits signals at 5.26 ppm (d, 3.6 Hz, α-anomer) and 4.68 ppm (d, 7.9 Hz, β-anomer) in a 38:62 ratio.

3. Results and discussion

3.1 Identification of S. mutans UA159 lipids: high performance thin-layer chromatography

Approximately 10-13 lipid components were resolved and detected by 2-D HPTLC of S. mutans UA159 cells grown to stationary-phase of culture growth (Fig. 1A). There were four lipid components that stained positive for sugars (Fig. 1B), one of which was also positive for phosphorus (Fig. 1C). Thus, these analyses established that this bacterium contained four glycolipids.

Fig. 1.

Fig. 1

Open in a new tab

Staining of Streptococcus mutans UA159 polar lipids isolated from stationary-phase cultures and separated by 2-D HPTLC. (A) General lipids, iodine. (B) Sugars, orcinol. (C) Phosphorus [12]. Four spots stained for sugars and one for both sugar and phosphorus.

2-D HPTLC analysis of the BHI extract showed no detectable spots on the plates indicating that the lipid components analyzed in S. mutans cell extracts did not originate from the culture medium. The S. mutans UA159 lipids identified in the present study are consistent with those reported by Cabacungan and Pieringer [1] who found similar lipid compositions in S. mutans BHT and FA-1 strains grown in a chemically defined medium.

3.2 Identification of S. mutans UA159 glycolipids: mass spectrometry

3.2.1 General

Spots identified as glycolipids were extracted from TLC plates and were directly nanosprayed into the instrument. Under the assumption that members within a glycolipid class differing only by the length of the acyl groups would ionize with similar efficiency this technique offers a facile comparison of the relative intensities of the various component members within a group. The analysis is however complicated by overlap between isotopic contributions that are isobaric at lower mass resolution. This necessitated that the data be acquired under the higher resolving power of the FT-ICR portion of the instrument (Fig. 2). For example diglucosyl monoacylglycerol (DGMAG) where the nominal masses for the octadecenyl and octadecanyl acyl groups are 703 and 705 respectively and denoted 18:1 and 18:0, the m+2 contribution for 18:1 resulting from two 13C or an 18O (and to a lesser extent 13C with 2H) overlaps with the nominal mass 18:0 component such that 57,000 (57K) resolving power at m/z 705 begins to suggest that at least two components are present (Fig. 2B). Resolving power of 113K clearly separates 18:1 from 18:0 components verifying the presence of 18:0 (Fig. 2C) and increasing the resolving power to 284K separated the various isotopic contributions for the m+2 component of 18:1 (Fig. 2D). Under collision-induced dissociation (CID) conditions, DGMAG loses a sugar group which will be discussed in detail below. The resulting MS/MS product ions show contributions from both 18:1 and 18:0 (Fig. 2E) in which m/z 541 is the nominal mass contribution for 18:1; m/z 542 is the 18:1 contribution still retaining one 13C atom; and the m/z 543 product ion has various isotopic contributions from both 18:1 and 18:0 components. These contributions are easily resolved and assigned with a resolving power of 294K (Fig. 2F). For this study, spectra were routinely acquired for full scans at a resolving power of 100K (m/z 400) and at 500K (m/z 400) under SIM conditions when better resolution was required.

Fig. 2.

Fig. 2

Open in a new tab

Overlap of diglycosylmonoacylglycerols with octadecenyl and octadecanyl acyl groups (18:1-DGMAG and 18:0-DGMAG) demonstrates the need for high resolution mass spectrometry. (A) Expanded region of the FT-ICR spectrum for the DGMAG sample. (B) Expansion of the m/z 705 region at 57K resolving power; the 18:1 and 18:0 components are barely distinguishable. (C) At 113K resolving power, the resolution is sufficient to clearly separate the 18:1 and 18:0 components thus confirming the presence of the 18:0 component. (D) At 284K resolving power, the multiple isotopic components contributing to the 18:1 m+2 ion are resolved. (E) Tandem mass spectrum of m/z 705 obtained in the linear ion trap expanding the mass region corresponding to C6H10O5 (sugar) loss; refer to text for details. (F) Transfer of m/z 705 product ions into the FT-ICR. Expansion of the m/z 543 product ion region obtained at 294K resolving power, distinguishing sugar loss from 18:0-DGMAG and 18:1-DGMAG still retaining various stable isotopes in the production.

For each glycolipid class a representation of the relative abundance of the various components contributing to the class is provided (Fig. 3). While not rigorously quantitative, this comparison still permits a useful survey of the major acyl contributors within each lipid class. To support this claim one must assume that the ionization and instrument transfer characteristics are not greatly influenced by the small differences in acyl chain length, generally spanning less than 200 Da for the group. Support that ionization is not significantly influenced by acyl length can be seen by comparing the positive and negative ion spectra of GPDGDAG, Figures 3D and 3E respectively. Although involving different solutions analyzed days apart, the sodiated adducts (most likely involving the sugar and carbonyl moieties) show a pronounced similarity to the anion pattern which is most likely centralizing the charge at the phosphate group. Both techniques represent ions differing greatly in type and charge local. Regarding ion transmission especially within a narrow mass range of less than 200 Da, the LTQ-FT™ utilizes multipoles which characteristically demonstrate a broad transfer range. In addition, such narrow ranges also do not exhibit “time-of-flight” effects between the linear ion trap and the FT-ICR; the LTQ-FT™ can trap ions an order of magnitude broader in range. It is informative to note that the largest acyl contributions for each glycolipid class investigated by MS were 16:0, 18:1, and 20:1. Quantitation of the fatty acid compositions of the major lipid classes in S. mutants UA159 by GLC confirmed that 16:0, 18:1 and 20:1 as major acyl groups (Custer, J.E., Goddard, B.D., Kaneshiro, E.S., in preparation). The observed acyl distribution extended as low as decyl (10:0 and 10:1) and as high as tetracosyl (24:0 and 24:1) with higher unsaturation observed with the longer, more abundant acyl groups (e.g. 18:3 and 20:3). Table 1 compliments Figure 3 providing accurate mass data confirming the elemental composition for each component. Tandem mass spectrometry data was used to determine the acyl distribution for each observed mass (see below). The use of “trace” for the acyl groups observed is typically used for acyl components that are under 2% of the intensity of the largest acyl component. Since the major acyl groups observed were 16:0 and 18:1, the parent ion containing these two groups was chosen as the representative example showing relative product ion distributions for each glycolipid class (Table 2).

Fig. 3.

Fig. 3

Open in a new tab

FT-ICR spectra for the glycolipids presented in this study. All are positive ion spectra of sodium adducts with the exception of the deprotonated, negative ion spectrum E. (A) Sodiated monoglucosyldiacylglycerol (MGDAG). (B) Sodiated diglucosyldiacylglycerol (DGDAG). (C) Sodiated diglucosylmonoacylglycerol (DGMAG). (D) Sodiated glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). (E) Deprotonated glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG).

Table 1.

Mass spectra of Streptococcus mutans UA159 glycolipids. Mass spectra are shown in Figure 3. See figure 11 for structures.

Observed Mass Elemental Composition Theoretical Mass Error (ppb) Major Acyl Groups Other Acyl Groups Observed
Monoglucosyldiacylglycerol (MGDAG)
669.45504 C35H66O10Na+ 669.45482 329 10:0/16:0; 14:0/12:0
695.47086 C37H68O10Na+ 695.47047 562 16:1/12:0; 16:0/12:1
697.48625 C37H70O10Na+ 697.48612 187 16:0/12:0
723.50199 C39H72O10Na+ 723.50177 305 16:0/14:1; 16:1/14:0 18:1/12:0
725.51762 C39H74O10Na+ 725.51742 276 16:0/14:0 18:0/12:0
749.51739 C41H74O10Na+ 749.51742 -40 16:1/16:1; 16:0/16:2 18:0/14:2; 18:2/14:0; 14:1/18:1
751.53319 C41H76O10Na+ 751.53307 160 16:0/16:1 18:1/14:0; 18:0/14:1; tra 20:1/12:0
753.54891 C41H78O10Na+ 753.54872 252 16:0/16:0 18:0/14:0; tr 20:0/12:0
777.54888 C43H78O10Na+ 777.54872 206 18:1/16:1 18:2/16:0; tr 14:1/20:1
779.56462 C43H80O10Na+ 779.56437 321 16:0/18:1 18:0/16:1; 20:1/14:0
781.58019 C43H82O10Na+ 781.58002 218 16:0/18:0
805.58032 C45H82O10Na+ 805.58002 372 18:1/18:1 20:1/16:1; 16:0/20:2; 18:0/18:2
807.59576 C45H84O10Na+ 807.59567 111 20:1/16:0 18:0/18:1
833.61151 C47H86O10Na+ 833.61132 228 18:1/20:1 16:0/22:2; ; tr 20:0/18:2; tr 18:0/20:2
835.62726 C47H88O10Na+ 835.62697 347 18:0/20:1 16:0/22:1; 18:1/20:0; tr 14:0/24.1
Diglucosyldiacylglycerol (DGDAG)
831.50781 C41H76O15Na+ 831.50764 201 10:0/16:0 14:0/12:0
857.52345 C43H78O15Na+ 857.52329 183 16:1/12:0 12:1/16:0
859.53904 C43H80O15Na+ 859.53894 113 16:0/12:0 tr 14:0/14:0; tr 18:0/10:0
883.53888 C45H80O15Na+ 883.53894 -71 16:0/14:2 16:1/14:1; tr 16:2/14:0
885.55461 C45H82O15Na+ 885.55459 19 16:0/14:1 16:1/14:0; 18:1/12:0
887.57047 C45H84O15Na+ 887.57024 256 16:0/14:0 18:0/12:0
911.57015 C47H84O15Na+ 911.57024 -102 16:1/16:1 16:0/16:2; 18:2/14:0; 18:0/14:2; 18:1/14:1
913.58607 C47H86O15Na+ 913.58589 194 16:0/16:1 18:1/14:0; 18:0/14:1; tr 20:1/12:0
915.60175 C47H88O15Na+ 915.60154 226 16:0/16:0 18:0/14:0; 20:0/12:0
937.58558 C49H86O15Na+ 937.58589 -334 18:2/16:1 18:1/16:2; 18:3/16:0; tr 20:1/14.2
939.60169 C49H88O15Na+ 939.60154 156 18:1/16:1 18:2/16:0; tr 20:1/14:1
941.61742 C49H90O15Na+ 941.61719 241 16:0/18:1 tr 18:0/16:1; tr 20:1/14:0
943.63307 C49H92O15Na+ 943.63284 240 18:0/16:0 tr 20:0/14:0
965.61727 C51H90O15Na+ 965.61719 79 18:2/18:1 tr 20:2/16:1; tr 20:1/16:2; tr 20:3/16:0
967.63299 C51H92O15Na+ 967.63284 152 18:1/18:1 20:1/16:1; 20:2/16:0
969.64873 C51H94O15Na+ 969.64849 244 20:1/16:0 18:0/18:1
971.66462 C51H96O15Na+ 971.66414 490 20:0/16:0 18:0/18:0
995.66403 C53H96O15Na+ 995.66414 -114 20:1/18:1
997.67985 C53H98O15Na+ 997.67979 57 18:0/20:1 12:0/18:1; tr 22:1/16:0
1023.69565 C55H100O15Na+ 1023.69544 202 20:1/20:1 22:1/18:1
1025.71028 C55H102O15Na+ 1025.71109 -793 20:0/20:1
Diglucosylmonoacylglycerol (DGMAG)
591.26227 C25H44O14Na+ 591.26233 -97 10:1
593.27811 C25H46O14Na+ 593.27798 224 10:0
617.27783 C27H46O14Na+ 617.27798 -239 12:2
619.29368 C27H48O14Na+ 619.29363 85 12:1
621.30938 C27H50O14Na+ 621.30928 165 12:0
645.30925 C29H50O14Na+ 645.30928 -42 14:2
647.32504 C29H52O14Na+ 647.32493 174 14:1
649.34067 C29H54O14Na+ 649.34058 142 14:0
673 C31H54O14Na+ 673.34058 too low 16:2
675.35639 C31H56O14Na+ 675.35623 240 16:1
677.37200 C31H58O14Na+ 677.37188 181 16:0
699.35618 C33H56O14Na+ 699.35623 -68 18:3
701.37198 C33H58O14Na+ 701.37188 146 18:2
703.38770 C33H60O14Na+ 703.38753 245 18:1
705.40329 C33H62O14Na+ 705.40318 159 18:0
729.40326 C35H62O14Na+ 729.40318 113 20:2
731.41896 C35H64O14Na+ 731.41883 181 20:1
733.43464 C35H66O14Na+ 733.43448 221 20:0
Glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG) - Positive Ions
1007.49305 C44H82O20PNa2+ 1007.49270 350 10:0/16:0 12:0/14:0
1033.50874 C46H84O20PNa2+ 1033.50835 380 16:1/12:0 12:1/16:0
1035.52432 C46H86O20PNa2+ 1035.52400 312 16:0/12:0 14:0/14:0
1059.52420 C48H86O20PNa2+ 1059.52400 191 16:1/14:1 16:0/14:2; 12:1/18:1; 14:0/16:2; 12:0/18:2
1061.54007 C48H88O20PNa2+ 1061.53965 398 16:0/14:1; 16:1/14:0 18:1/12:0; 12:1/18:0
1063.55561 C48H90O20PNa2+ 1063.55530 294 16:0/14:0 18:0/12:0
1087.55576 C50H90O20PNa2+ 1087.55530 425 16:1/16:1 18:1/14:1; 16:0/16:2; 18:2/14:0; tr 20:1/12:1
1089.57131 C50H92O20PNa2+ 1089.57095 333 16:0/16:1 18:1/14:0; tr 18:0/14:1
1091.58693 C50H94O20PNa2+ 1091.58660 304 16:0/16:0 18:/14:0
1115.58701 C52H94O20PNa2+ 1115.58660 370 18:1/16:1 18:2/16:0; tr 20:1/14:1
1117.60254 C52H96O20PNa2+ 1117.60225 261 18:1/16:0 18:0/16:1; 20:1/14:0
1119.61832 C52H98O20PNa2+ 1119.61790 377 18:0/16:0 20:0/14:0; tr 12:0/22:0
1143.61841 C54H98O20PNa2+ 1143.61790 448 18:1/18:1 20:1/16:1; 20:2/16:0
1145.63396 C54H100O20PNa2+ 1145.63355 360 20:1/16:0 18:0/18:1
1147.64967 C54H102O20PNa2+ 1147.64920 411 20:0/16:0 18:0/18:0; 22:0/14:0; tr 24:0/12:0
1169.63404 C56H100O20PNa2+ 1169.63355 421 20:2/18:1 22:3/16:0; 18:2/20:1
1171.64970 C56H102O20PNa2+ 1171.64920 429 20:1/18:1 tr 22:2/16:0
1173.66537 C56H104O20PNa2+ 1173.66485 445 20:0:18:1; 18:0/20:1 22:0/16:1; 22:1/16:0
Glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG) - Negative Ions
961.51400 C44H82O20P- 961.51426 -265 10:0/16:0
987.52973 C46H84O20P- 987.52991 -177 16:1/12:0; 16:0/12:1
989.54529 C46H86O20P- 989.54556 -268 16:0/12:0 tr 14:0/14:0
1013.54540 C48H86O20P- 1013.54556 -153 16:1/14:1 16:0/14:2
1015.56099 C48H88O20P- 1015.56121 -212 16:1.14:0; 16:0/14:1 18:1/12:0
1017.57669 C48H90O20P- 1017.57686 -163 16:0/14:0 18:0/12:0
1041.57658 C50H90O20P- 1041.57686 -264 16:1/16:1 18:1/14:1
1043.59243 C50H92O20P- 1043.59251 -72 16:0/16:1 18:1/14:0; tr 18:0/14:1
1045.60802 C50H94O20P- 1045.60816 -130 16:0/16:0 18:0/14:0
1069.60795 C52H94O20P- 1069.60816 -192 18:1/16:1 18:2/16:0; tr 20:1/14:1
1071.62350 C52H96O20P- 1071.62381 -285 18:1/16:0 18:0/16:1; tr 20:1/14:0
1073.63930 C52H98O20P- 1073.63946 -145 18:0/16:0 20:0/14:0
1097.63935 C54H98O20P- 1097.63946 -96 18:1/18:1 20:1/16:1; 20:2/16:0
1099.65500 C54H100O20P- 1099.65511 -96 20:1/16:0 18:0/18:1
1101.67103 C54H102O20P- 1101.67076 249 20:0/16:0 18:0/18:0
1123.65492 C56H100O20P- 1123.65511 -165 too lowa
1125.67051 C56H102O20P- 1125.67076 -218 20:1/18:1 tr 22:2/16:0
1127.68625 C56H104O20P- 1127.68641 -138 18:0/20:1; 20:0/18:1 tr 22:1/16:0; tr 16:1/22:0
Open in a new tab
a

The ion was too low to be observed in the FT-ICR and determine an accurate mass and the mass error (error relative to the theoretical mass).

Table 2.

Product ions of glycolipids from Streptococcus mutans UA159. Structures are shown in Figure 11.

Product Ion Relative Abundance Accurate Mass Elemental Composition Error (ppb) Description of loss
MGDAG MS2: C43H80O10Na+ (779)
523 100 523.32427 C27H48O8Na+ 248 16:0 acid
497 75.1 497.30862 C25H46O8Na+ 261 18:1 acid
617 9.2 617.51172 C37H70O5Na+ 280 sugar (C6H10O5)
495 8.9 495.29293 C25H44O8Na+ 182 18:0 acid
525 4.0 525.33998 C27H50O8Na+ 361 16:1 acid
469 1.8 469.27721 C23H42O8Na+ 43 20:1 acid
551 1.1 551.35508 C29H52O8Na+ -654 14:0 acid (buried)
DGDAG MS2: C49H90O15Na+ (941)
685 100 685.37729 C33H58O13Na+ 476 16:0 acid
659 88.7 659.36160 C31H56O13Na+ 434 18:1 acid
779 22.6 779.56477 C43G80O10Na+ 512 sugar (C6H10O5)
657 8.6 657.34586 C31H54O13Na+ 299 18:0 acid
523 6.2 523.32439 C27H48O8Na+ 477 16:0 acid & sugar
497 5.5 497.30878 C25H46O8Na+ 583 18:1 acid & sugar
687 4.1 687.39295 C33H60O13Na+ 489 16:1 acid
631 1.9 631.33033 C29H52O13Na+ 501 20:1 acid
713 1.0 713.40886 C35H62O13Na+ 836 14:0 acid
495 0.6 495.29242 C25H44O8Na+ -848 18:0 acid & sugar
525 0.3 too lowa too low too low 16:1 acid & sugar
617 0.3 too low too low too low both sugars
551 0.2 too low too low too low 14:0 acid & sugar
469 0.1 too low too low too low 20:1 acid & sugar
DGMAG MS2: C31H58O14Na+ (677 - contains 16:0)
515 100 515.31902 C25H48O9Na+ -68 sugar (C6H10O5)
353 1.8 353.26610 C19H38O4Na+ -372 both sugars
659 0.8 too low too low too low water
421 0.6 too low too low too low 16:0 acid
347 0.6 too low too low too low B2 - H sugar ionb
365 0.3 too low too low too low C2 + H sugar ionb
259 0.2 too low too low too low sugar & 16:acid
DGMAG MS3: C25H48O9Na+ (515 from 677 - contains 16:0)
353 100 353.26617 C19H38O4Na+ -173 sugar (C6H10O5)
352 4.6 too low too low too low sugar w/o H-transfer
497 2.4 too low too low too low water
185 1.7 too low too low too low monoacylglycerol
259 1.7 too low too low too low 16:0 acid
DGMAG MS2: C33H60O14Na+ (703 - contains 18:1)
541 100.0 541.33435 C27H50O9Na+ -656 sugar (C6H10O5)
379 2.0 379.28181 C21H40O4Na+ -188 both sugars
685 0.8 too low too low too low water
365 0.6 too low too low too low C2 + H sugar ionb
347 0.5 too low too low too low B2 - H sugar ionb
421 0.3 too low too low too low 18:1 acid
259 0.1 too low too low too low sugar & 18:1 acid
DGMAG MS3: C27H50O9Na+ (541 from 703 - contains 18:1)
379 100.0 379.28179 C21H40O4Na+ -241 sugar (C6H10O5)
523 1.5 too low too low too low water
185 0.9 too low too low too low monoacylglycerol
378 0.6 too low too low too low sugar w/o H-transfer
259 0.5 too low too low too low 18:1 acid
GPDGDAG MS2: C52H96O20PNa2+ (1117)
941 100 941.61733 C49H99O15Na+ 144 C3H6O5PNa
923 4.7 923.60710 C49H88O14Na+ 509 C3H8O6PNa
835 4.4 835.34687 C34H62O18PNa2+ 598 18:1 acid
861 4.3 861.36250 C36H64O18PNa2+ 557 16:0 acid
659 4.2 659.36158 C31H56O13Na+ 404 C3H6O5 PNa & 18:1 acid
685 3.9 685.37725 C33H58O13Na+ 418 C3H6O5PNa & 16:0 acid
1025 0.9 1025.55553 C49H88O17PNa2+ 608 C3H8O3
1043 0.8 1043.56605 C49H90O18PNa2+ 555 C3H6O2
833 0.4 833.33123 C34H60O18PNa2+ 612 18:0 acid
657 0.4 657.34599 C31H54O13Na+ 497 C3H6O5PNa & 18:0 acid
863 0.1 too low too low too low 16:1 acid
807 0.1 too low too low too low 20:1 acid
889 0.1 too low too low too low 14:0 acid
687 0.1 too low too low too low C3H6O5PNa & 16:1 acid
631 0.1 too low too low too low C3H6O5PNa & 20:1 acid
731 0.0 too low too low too low C3H6O5PNa & 14:0 acid
GPDGDAG MS3: C49H90O15Na+ (941 from 1117)
659 100 659.36108 C31H56O13Na+ -354 18:1 acid
685 97.3 685.37672 C33H58O13Na+ -356 16:0 acid
779 25.5 779.56410 C43H80O10Na+ -347 sugar (C6H10O5)
657 8.1 657.34544 C31H54O13Na+ -340 18:0 acid
497 6.4 497.30843 C25H46O8Na+ -121 18:1 acid & sugar
523 6.1 523.32405 C27H48(O)8Na+ -172 16:0 acid & sugar
687 3.6 687.39225 C33H60O13Na+ -529 16:1 acid
631 2.5 631.32999 C29H52O13Na+ -37 20:1 acid
713 1.2 713.40787 C35H62O13Na+ -552 14:0 acid
495 0.6 too low too low too low 18:0 acid & sugar
617 trace - buried 617.51114 C37H70O5Na+ -659 both sugars
525 trace too low too low too low 16:1 acid & sugar
469 trace too low too low too low 20:1 acid & sugar
551 trace too low too low too low 14:0 acid & sugar
GPDGDAG MS2: C52H96O20P- (1071)
997 100 997.58818 C49H90O18P- 1154 C3H6O2
789 64.3 789.36863 C34H62O18P- 890 18:1 acid
815 63.7 815.38418 C36H64O18P- 739 16:0 acid
833 32.9 833.39480 C36H66O19P- 789 16:0 ketene
807 32.3 807.37925 C34H64O19P- 938 18:1 ketene
315 21.3 315.04884 C9H16O10P- 575 glucosyldiacylglycerol
979 6.4 979.57732 C49H88O17P- 874 C3H8O3
787 5.4 787.35271 C34H60O18P- 549 18:0 acid
459 4.4 459.09132 C15H24O14P- 876 C3H6O2 & both acids
715 4.4 715.33182 C31H56O16P- 939 C3H6O2 & 18:1 acid
741 4.2 741.34738 C33H58O16P- 785 C3H6O2 & 16:0 acid
477 3.6 477.10124 C15H28O15P- -508 C3H6O2 & acid & ketene
759 2.8 759.35756 C33H60O17P- 260 C3H6O2 & 16:0 ketene
733 2.5 733.34181 C31H58O17P- 133 C3H6O2 & 18:1 ketene
817 2.2 too low too low too low 16:1 acid
835 2.0 too low too low too low 16:1 ketene
533 1.8 533.12745 C18H30O16P- -464 both acids
805 1.6 805.36302 C34H62O19P- 221 18:0 ketene
761 1.4 too low too low too low 20:1 acid
551 0.8 too low too low too low acid & ketene
843 0.7 too low too low too low 14:0 acid
861 0.6 too low too low too low 14:0 ketene
495 0.4 too low too low too low C3H6O2 & both ketenes
779 0.4 too low too low too low 20:1 ketene
713 0.3 too low too low too low C3H6O2 & 18:0 acid
743 trace too low too low too low C3H6O2 & 16:1 acid
687 trace too low too low too low C3H6O2 & 20:1 acid
769 trace too low too low too low C3H6O2 & 14:0 acid
731 trace too low too low too low C3H6O2 & 18:0 ketene
705 trace too low too low too low C3H6O2 & 20:1 ketene
Open in a new tab
a

too low: the product ion from collision induced dissociation of the parent ion given in bold for each section, was too low to be observed in the FT-ICR and thus determine an accurate mass which would have also generated an elemental composition and mass error value.

b

Not losses but sodiated glycoconjugates according to the nomenclature designated by Domon and Costello [14].

3.2.2 Monoglucosyldiacylglycerol and diglucosyldiacylglycerol

Sodium adducts of monoglucosyldiacylglycerol (MGDAG) and diglucosyldiacylglycerol (DGDAG) form similar collision-induced dissociation (CID) profiles. An example of each is given in Table 2 with 16:0 and 18:1 as the two acyl groups. The fragmentation is dominated by acid loss as illustrated in (Fig. 4A); acid loss from the sn-2 position can also be observed in a similar manner. The other major fragmentation pathway involves hydrogen transfer and loss of the sugar moiety resulting in a characteristic Y-ion for glycoconjugates as designated by Domon and Costello ([17].

Fig. 4.

Fig. 4

Open in a new tab

Proposed mechanisms consistent with accurate mass product ion data and similar to other reported mechanisms. A. Mechanism common for carboxylic acid loss from glycolipids similar to Scheme 2 in Guella et al. [18]. B. Mechanism accounting for the C3H6O5PNa losses observed in the sodiated GPDGDAG product ion spectra. C. Mechanism accounting for the prevalent C3H6O2 losses observed in the deprotonated GPDGDAG product ion spectra similar to Scheme 4 in P.F. James, et. al. [17].

The MGDAG components are summarized (Table 1), which can be used with data in Figure 3A to show relative abundance of the various components in this lipid class. There are other components present including a significant number of oxidation products. Although effort was made to prevent oxidation of the sample, it is presently unknown whether the oxidation products originated from the organism or due to sample handling. Collision-induced dissociation was performed on each species listed in Table 1 to ensure that these components are MGDAG and to determine their acyl distributions. The most predominant product ions correspond to acid losses (the larger acid loss is listed first in the pair). Other observable but less abundant acyl pairs are listed in the table in the order of relative abundance. Acid loss has been shown to favor the sn-1 position for sodiated mono- and digalactosyldiacylglycerols [18]. In the present study, C6H10O5 loss from the sugar portion of the molecule was also observed.

3.2.3 Diglucosylmonoacylglycerol

While characterizations of diglucosylmonoacyloglycerols (DGMAG) are rare in the literature, examples have been reported. Two possible basic structures are possible with either the two sugars linked together or separately attached to the glycerol portion (Fig. 5). It has been reported that the cyanobacterium, Synechocystis, contains a DGMAG similar to that shown in Figure 5A except that the sugar linkage is 1,6 with a palmitoyl substitution at the terminal sugar’s C-6 position; it also has the acyl group substituted at the sn-2 position [19]. Colebroside A is an example with structure similar to that shown in Figure 5B [20]. The present report describes the first observation and tandem mass spectrometry for unsubstituted DGMAG, which is a minor glycolipid found in S. mutans UA159. NMR (see below) clearly indicates that the sugars are linked together with a 1,2’- α-linkage and that acylation of glycerol is at the sn-1 position (Fig. 5A). Even though the diacyl glycolipids DGDAG and MGDAG show loss of the sugar moiety, acid loss is the major product ion observed. In contrast DGMAG showed almost exclusive sugar loss in both the MS2 and MS3 experiments (Table 2). The fact that acid loss is observed two orders of magnitude lower in product ion abundance may be indicative of a stronger sodium ion preference for the carbonyl group thus hindering proton transfer and subsequent acid loss. Although present <1% relative abundance in MS2 spectra (Fig. 6), there are two product ions that also support the linked sugar structure shown in Figure 5A as opposed to the separated sugar structure of Figure 5B. These product ions are B2 as a sodium adduct with a H transfer to the glycerol (m/z 347) and C2 as a sodium adduct with a H-transfer from the glycerol (m/z 365), using the ion nomenclature designated by Domon and Costello [17].

Fig. 5.

Fig. 5

Open in a new tab

Possible structures for diglucosylmonoacylglycerol (DGMAG). (A) Glucose rings linked together; the DGMAG observed in this study has this structure with the 1-2’ linkage and the sn-1 acylation as shown. (B) Glucose rings separated; this structure has also been observed for other bacteria (see text) and was not observed in S. mutans UA159 DGMAG.

Fig. 6.

Fig. 6

Open in a new tab

Product ion spectrum (LTQ) of the DGMAG parent containing an octyldecenyl acyl (18:1) group, m/z 703. While the product ions are dominated by sugar loss, two small ions labeled B2 and C2 support the linked sugar structure shown in Figure 5A (refer to text).

3.2.4 Glycerophosphoryldiglucosyldiacylglycerol

In glycerophosphoryldiglucosyldiacylglycerides (GPDGDAG) the diacylglyceride is not bound through a phosphate group to the sugar. While there are many studies of phosphatidylglucosides especially regarding lipid rafts and other lipid domains [21, 22] GPDGDAG has received considerably less attention. GPDGDAG has been identified in Acholepasma laidlawii [23--26] by NMR but without mass spectrometry data. Also, GPDGDAG had been tentatively identified in another strain of S. mutans but MS was not performed [1].

CID of the sodium adduct is dominated by hydrogen transfer to the sugar followed by loss of C3H6O5PNa to produce a product ion similar in structure to the parent ions of DGDAG (Fig. 4B). This is further supported by MS3; comparison of the m/z 941 product ion from m/z 1117 for GPDGDAG (Table 2) with the MS2 of the m/z 941 parent for DGDAD. Not only are the observed losses identical but the relative intensities are also strongly similar. MS3 generates strong acid losses and is used in preference to MS2 in determination of the acyl components.

In addition to the sodium ion adducts similar to the glycolipids, the phosphate group contributes to intense anions that are also useful in the identification and characterization of GPDGDAG. The major product ion observed for the GPDGDAG anions was C3H6O2 loss most likely directly involving the charge site (Fig. 4C). The anions also show strong acid and ketene losses characteristic of phosphatidyl lipids but remote from the charge site. The acid and ketene losses can also be used to determine the acyl components. The major product ions obtained through low energy CID of these anion components in the ion trap, presents very little evidence regarding the sugar structure with the exception of m/z 315 (Fig. 7). This ion has an elemental composition suggesting a B1 ion that also contains a phosphate group with an unsubstituted glyceryl; the positive ion fragmentation supports the glycerol molecule attached to the phosphate group which is in agreement with the NMR data (see below).

Fig. 7.

Fig. 7

Open in a new tab

Proposed structure for the m/z 315 product ion arising from glucosyldiacylglycerol loss from deprotonated GPDGDAG. The presence of this ion supports the glycerophosphoryl group being attached to a different glucose than the diacylglycerol.

3.3 Identification of S. mutans UA159 glycolipids: nuclear magnetic resonance spectroscopy

3.3.1 Monoglucosyldiacylglycerol (MGDAG)

This fraction was characterized by 1H-NMR and COSY using CDCl3 as the solvent (Table 3). The linkage of the sugar was shown to be alpha by the small coupling constant of the anomeric signal (3.6 Hz). Positions 4-6 of the sugar were difficult to assign because of overlapping signals. Assignment of the sugar as glucose was determined by hydrolysis with 2 M D2SO4/D2O at 100 °C for 1 h, followed by direct 1H-NMR measurement.

Table 3.

1H-NMR analysis of S. mutans UA159 glycolipids. See figure 11 for structures and desgination of positions.

Position 1H-NMR chemical shifts Hauksson et al. [13]
Monoglucosyldiacylglycerol (MGDAG)
 Glucose, 1 4.86 ppm (d, 3.6 Hz)
 Glucose, 2 3.52 ppm (dd, 3.6, 9.6 Hz)
 Glucose, 3 3.76 ppm (t, 9.4 Hz)
 Glyceryl, 1 4.11 ppm (dd, 5.8, 11.6 Hz)
 Glyceryl, 1’ 4.37 ppm (dd, 3.9, 11.6 Hz)
 Glyceryl, 2 5.23 ppm (m)
 Glyceryl, 3 3.84 ppm*
 Glyceryl, 3’ 3.61 ppm*
Diglucosyldiacylglyerol (DGDAG)
 Glucose A, 1 4.81 ppm (d, 3.5 Hz)
 Glucose A, 2 3.41 ppm*
 Glucose B, 1 4.78 ppm (d, 3.7 Hz)
 Glucose B, 2 3.27 ppm (dd, 3.6, 9.5 Hz)
 Glyceryl, 1 4.26 ppm (dd, 3.2, 12.0 Hz)
 Glyceryl, 1’ 4.02 ppm (dd, 6.5, 12.0 Hz)
 Glyceryl, 2 5.05 (m)
 Glyceryl, 3 3.67 ppm*
 Glyceryl, 3’ 3.47 ppm (dd, 5.2, 10.9 Hz)
Diglucosylmonoacylglycerol (DGMAG)
 Glucose A, 1 4.92 ppm (d, 3.4 Hz)
 Glucose A, 2 3.35 ppm*
 Glucose B, 1 4.83 ppm (d, 3.6 Hz)
 Glucose B, 2 3.18 ppm (dd, 3.6, 9.5 Hz)
 Glyceryl, 1 3.96 ppm*
 Glyceryl, 1’ 4.03 ppm*
 Glyceryl, 2 3.85 ppm*
 Glyceryl, 3 3.34 ppm*
 Glyceryl, 3’ 3.57 ppm*
Glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG)
 Glucose A, 1 4.92 ppm (d, 3.7 Hz) 4.93 ppm
 Glucose A, 2 3.36 ppm* 3.38 ppm
 Glucose B, 1 4.82 ppm (d, 4.2 Hz) 4.84 ppm
 Glucose B, 2 3.20 ppm* 3.23 ppm
 Glyceryl A, 1 4.35 ppm* 4.39 ppm
 Glyceryl A, 1’ 4.16 ppm* 4.15 ppm
 Glyceryl A, 2 5.11 ppm* 5.14 ppm
 Glyceryl A, 3 3.57 ppm* 3.59 ppm
 Glyceryl A, 3’ 3.70 ppm* 3.71 ppm
 Glyceryl B, 1, 1’ 3.28 ppm* 3.31 ppm
 Glyceryl B, 2 3.49 ppm* 3.52 ppm
 Glyceryl B, 3, 3’ 3.65 ppm* 3.67 ppm
Open in a new tab
**

Chemical shifts determined by COSY.

3.3.2 Diglucosyldiacylglycerol (DGDAG)

This fraction was characterized by 1H-NMR, COSY, TOCSY and NOESY using CDCl3 /d4-CH3OH 3:1 as the solvent (Fig. 8). The linkages of the sugars were shown to both be alpha by the small coupling constants of the anomeric signals (ca. 3.6 Hz). The 1,2’-linkage of the sugars was determined by NOESY which showed a cross peak between 4.78 and 3.41 ppm. This was confirmed by 1D ROESY. Linkage of the other sugar and the glyceryl backbone was evident from a cross peak between 4.81 and 3.47 ppm by NOESY. Assignment of the sugar as glucose was determined by hydrolysis with 2 M D2SO4/D2O at 100 °C for 1 h, followed by direct 1H-NMR measurement (Fig. 9).

Fig. 8.

Fig. 8

Open in a new tab

COSY 1H-NMR spectrum of diglycosyl diacylglycerol. Solvent: CDCl3 /d4-CH3OH 3:1.

Fig. 9.

Fig. 9

Open in a new tab

Sugar analysis by 1H-NMR. A. Anomeric region of the hydrolysate of diglucosyl diacylglycerol;. B. Anomeric region of glucose. Solvent: 2M D2SO4/D2O.

3.3.3 Diglycosylmonoacylglycerol (DGMAG)

This fraction was characterized by 1H-NMR and COSY using d6-DMSO containing 2% D2O as the solvent. The signals of the glyceryl group were detected by COSY. The glyceryl group was determined to be acylated at position-1 (sn-1) by the higher chemical shifts of the corresponding signals.

The linkages of the sugars were shown to be alpha by the small coupling constants of the anomeric signals (ca. 3.5 Hz). The 1H-NMR signals of the C2 positions of the sugars were evident from their correlation to the signals of the anomeric H-atoms by COSY. The 1,2’-linkage of the sugars was determined by 1D ROESY. Irradiation of the anomeric signal at 4.83 led to enhancement of a signal at 3.35 ppm (the C2 H-atom of the other sugar) as well as the signal of the coupled C2 H-atom. When applied to the anomeric signal at 4.92 ppm, 1D ROESY did not enhance the signal of C2 H-atom at 3.18 ppm. Because signal of the C2 H-atom at 3.35 ppm was overlapping with other signals, the outcome of the 1D ROESY experiments was confirmed using CDCl3 /d4-CH3OH 3:1 as the solvent. In this solvent, the anomeric signals at 4.84 ppm (d, 3.2 Hz) and 4.81 ppm (d, 3.8 Hz) were shown by COSY to be coupled to signals of C2 H-atoms at 3.46 and 3.30 ppm, respectively, while NOE was confirmed by 1D ROESY between the 4.81 and 3.46 ppm signals.

The assignment of the sugar groups as glucose was determined by hydrolysis with 2M D2SO4/D2O at 100 °C for 1 h, followed by direct 1H-NMR measurement.

3.3.4 Glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG)

This fraction was characterized by 1H-NMR and COSY using d6-DMSO containing 2% D2O as the solvent. The spectra correlated well with those of compound 1 described by Hauksson et al. [23] (Fig. 10).

Fig. 10.

Fig. 10

Open in a new tab

COSY 1H-NMR spectrum of glycerophosphoryl diglucosyl diacylglycerol. Solvent: d6-DMSO + 2% D2O.

3.4 S. mutans glycolipids

The abundance of glycolipids and their accumulation with culture age in S. mutans is correlated with the acidification as well as depletion of nutrients in the culture medium suggests that these compounds participate in adhesion and inclusion in teeth biofilms [27]. Although glycolipids and/or other lipids might confer greater resistant to acidic conditions, there is currently insufficient evidence showing that these compounds specifically serve that function in S. mutans.

Other strains of S. mutans were shown to synthesize these glycolipids [1] and the present study on UA159 provides detailed MS and NMR confirming the nature of their structures. Glucosyltransferases have been identified on the S. mutans surface and in the extracellular milieu released by the bacteria [4-6, 25]. Hence it is possible, that the formation of these glycolipids first involves de novo synthesis of glycerolipids (i.e.,PG) followed by remodeling by the additions of glucose to PG at the cell surface and/or in the extracellular environment. This would be consistent with previous observations of lipid synthesis in or by this organism and the presence of these glycolipids in the culture medium [1, 2].

Since sucrose is a particularly cariogenic disaccharide and can either be taken up by S. mutans cells or hydrolyzed extracellularly [29-31], it might serve as a source of glucose molecules that are transferred to PG thus giving rise to these glycolipids. Future studies might include testing whether incubation of PG with “conditioned” media in which S. mutans was grown and presumably containing the necessary enzymes, leads to the production of these same glycolipids.

4. Conclusions

Three glycolipids and one phosphoglycolipid were detected in the oral pathogen Streptococcus mutans strain UA159 grown in standing cultures to late stationary phase. Definitive structural identities were obtained by specific staining of 2-D high-performance thin-layer chromatography plates, high-resolution mass spectrometric analyses and nuclear magnetic resonance spectroscopy. The glycolipids included monoglucosyldiacylglycerol, diglucosyldiacylglycerol, diglucosylmonoacylglycerol, and glycerophosphoryldiglucosyldiacylglycerol (Fig. 11). The two glucose moieties in the later three compounds were connected via an α-1,2 glycosidic linkage. Galactosyl lipids were not detected as reported in S. mutans strain FA-1 [32]. We hypothesize that some phosphatidylglycerol molecules synthesized de novo are modified by extracellular or externally located cellular glucosyltransferases to form these glycolipids found associated with the cell as well as in the culture medium. More research is required to determine the role of these compounds in the physiology of S. mutans, whether they are involved in enabling the bacterium to live and proliferate under changing environmental conditions and whether these glycolipids play a role in oral disease.

Fig. 11.

Fig. 11

Open in a new tab

Structures of Streptococcus mutans UA159 glycolipids.

Highlights.

  • Larry Sallans BBA

  • Streptococcus mutans UA159 synthesizes four glycolipids.

  • Definitive structural identities were determined by HPTLC, MS and NMR

  • S. mutans glycolipids were comprised of glucosyl, not galactosyl moeities.

  • This is the first observation of unsubstituted DGMAG in any organism.

  • These glycolipids might enhance biofilm formation involved in dental pathogenesis

Acknowledgments

We thank Bryan Goddard for helpful comments on the manuscript. This study was supported in part by NIH grants RO1 AI064084 and NIH RR19900.

Abbreviations

BHI

brain-heart infusion

BHT

butylated hydroxytoluene

CID

collision-induced dissociation

CL

cardiolipin, diphosphatidylglycerol

COSY

correlation spectroscopy

DAG

diacylglycerol

ddH2O

double-distilled water

DGDAG

diglucosyldiacylglycerol

DGMAG

diglucosylmonoacylglycerol

DMSO

dimethylsulfoxide

FT-ICR

Fourier transform ion cyclotron resonance

GPDGDAG

glycerophosphoryldiglucosyldiacylglycerol

HPTLC

high performance thin-layer chromatography

MS

mass spectrometry

MGDAG

monoglucosyldiacylglycerol

NMR

nuclear magnetic resonance

NOESY

nuclear Overhauser effect spectroscopy

PG

phosphatidylglycerol

ROESY

rotating frame nuclear Overhauser effect spectroscopy

TLC

thin-layer chromatography

TOCSY

Total correlation spectroscopy

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Contributor Information

Larry Sallans, Email: Larry.Sallans@uc.edu.

José-Luis Giner, Email: jlginer@syr.edu.

David J. Kiemle, Email: dkiemle@esf.edu.

Jenny E. Custer, Email: custerje@mail.uc.edu.

References

  • 1.Cabacungan E, Pieringer RA. Excretion of extracellular lipids by Strept ococcus mutans BHT and FA-1. Infect Immun. 1980;27:556–562. doi: 10.1128/iai.27.2.556-562.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Cabacungan E, Gustow E, Pieringer RA. In vivo studies on stereospecificity of the monoglyceride kinase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase and CDP-diglyceride synthase of Streptococcus mutans BHT using the stereoisomers of the ether lipid, dodecylglycerol. Biochim Biophys Acta. 1988;962:241–247. doi: 10.1016/0005-2760(88)90166-x. [DOI] [PubMed] [Google Scholar]
  • 3.Ajdić D, McShan WM, McLaughlin RE, Savić G, Chang J, Carson MB, Primeaux C, Tian R, Kenton S, Lin HS, Qian Y, Li S, Zhu H, Najar F, Lai H, White J, Roe BA, Ferretti JJ. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci USA. 2002;99:14434–14439. doi: 10.1073/pnas.172501299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986;50:353–380. doi: 10.1128/mr.50.4.353-380.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Hamada S, Slade HD. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Mol Biol Rev. 1980;44:331–384. doi: 10.1128/mr.44.2.331-384.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.van Hoogmoed CG, van der Kuijl-Booij M, van der Mei HC, Busscher HJ. Inhibition of Streptococcus mutans NS adhesion to glass with and without salivary conditioning film by biosurfactant-releasing Streptococcus mitis strains. Appl Environ Microbiol. 2000;66:659–663. doi: 10.1128/aem.66.2.659-663.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Quivey RG, Jr, Faustoferri R, Monahan K, Marquis R. Shifts in membrane fatty acid profiles associated with acid adaptation of Streptococcus mutans. FEMS Microbiol Lett. 2000;189:89–92. doi: 10.1111/j.1574-6968.2000.tb09211.x. [DOI] [PubMed] [Google Scholar]
  • 8.Fozo EM, Quivey RG. Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments. Appl Environ Microbiol. 2003;70:929–936. doi: 10.1128/AEM.70.2.929-936.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Fozo EM, Quivey RG. The fabM gene product of Streptococcus mutans is responsible for the synthesis of monounsaturated fatty acids and is necessary for survival at low pH. J Bacteriol. 2004;186:4152–4158. doi: 10.1128/JB.186.13.4152-4158.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Fozo EM, Kajfasz JK, Quivey RG., Jr Low pH-induced membrane fatty acid alterations in oral bacteria. FEMS Microbiol Lett. 2004;238:291–295. doi: 10.1016/j.femsle.2004.07.047. [DOI] [PubMed] [Google Scholar]
  • 11.Fozo EM, Scott-Anne K, Koo H, Quivey RG., Jr Role of unsaturated fatty acid biosynthesis in virulence of Streptococcus mutans. Infect Immun. 2007;75:1537–1539. doi: 10.1128/IAI.01938-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  • 13.Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226:497–509. [PubMed] [Google Scholar]
  • 14.Dittmer JC, Lester RL. A simple, specific spray for the detection of phospholipids on thin-layer chromatograms. J Lipid Res. 1964;15:126–127. [PubMed] [Google Scholar]
  • 15.Kiemle DJ, Stipanovic AJ, Mayo KE. Proton NMR methods in compositional characterization of polysaccharides. ACS Symposium Series. 200;864:122–139. [Google Scholar]
  • 16.James PF, Perugini MA, O’Hair RAJ. Sources of artifacts in the electrospray ionization mass spectra of saturated diacylglycerophosphocholines:from condensed phase hydrolysis reactions through to gas phase intercluster reactions. J Am Soc Mass Spectrom. 2006;17:384–394. doi: 10.1016/j.jasms.2005.11.009. [DOI] [PubMed] [Google Scholar]
  • 17.Domon B, Costello CE. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconjugate J. 1988;5:397–409. [Google Scholar]
  • 18.Guella G, Frassanito R, Mancini I. A new solution for an old problem: the regiochemical distribution of the acyl chains in galactolipids can be established by electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2003;17:1982–1994. doi: 10.1002/rcm.1142. [DOI] [PubMed] [Google Scholar]
  • 19.Kim YH, Choi K-S, Hong J, Yoo JS, Kim MS. Identification of acylated glycoglycerolipids from a cyanobacterium, Synechocystis sp. by tandem mass spectrometry. Lipids. 1999;34:847–853. doi: 10.1007/s11745-999-0432-2. [DOI] [PubMed] [Google Scholar]
  • 20.Yang H, Jiang B, Hou A-J, Lin Z-W, Sun H-D. Colebroside A, a new diglucoside of fatty acid ester of glycerin from Clerodendrum cobrookianum. J Asian Nat Prod Res. 2000;2:177–185. doi: 10.1080/10286020008039909. [DOI] [PubMed] [Google Scholar]
  • 21.Nagatsuka Y, Hirabayashi Y. Phosphatidylglucoside: A new marker for lipid rafts. Biochim Biophys Acta. 2008;1780:405–409. doi: 10.1016/j.bbagen.2007.08.016. [DOI] [PubMed] [Google Scholar]
  • 22.Murate M, Hayakawa T, Ishii K, Inadome H, Greimel P, Watanabe M, Nagatsuka Y, Ito K, Ito Y, Takahashi H, Hirabayashi Y, Kobayashi T. Phosphatidylglucoside forms specific lipid domains on the outer leaflet of the plasma membrane. Biochemistry. 2010;49:4732–4739. doi: 10.1021/bi100007u. [DOI] [PubMed] [Google Scholar]
  • 23.Hauksson JB, Lindblom G, Rilfors L. Structures of glucolipids from the membrane of Acholeplasma laidlawii Strain A-EF22.1. Glycerophosphoryldiglucosyldiacylglycerol and Monoacylbisglycerophosphoryldiglucosyldiacylglycerol. Biochim Biophys Acta. 1994;1214:124–130. doi: 10.1016/0005-2760(94)90035-3. [DOI] [PubMed] [Google Scholar]
  • 24.Danino D, Kaplun A, Lindblom G, Rilfors L, Orädd G, Hauksson JB, Talmon Y. Cryo-TEM and NMR studies of a micelle-forming phosphoglucolipid from membranes of Acholeplasma laidlawii A and B. Chem Phys Lipids. 1997;85:75–89. doi: 10.1016/s0009-3084(96)02640-0. [DOI] [PubMed] [Google Scholar]
  • 25.Andersson AS, Rilfors L, Lewis RN, McElhaney RN, Lindblom G, Lindblom G. Occurrence of monoacyl-diglucosyl-diacyl-glycerol and monoacyl-bis-glycerophosphoryl-diglucosyl-diacyl-glycerol in membranes of Acholeplasma laidlawii Strain B-PG9. Biochim Biophys Acta. 1998;1389:43–49. doi: 10.1016/s0005-2760(97)00091-x. [DOI] [PubMed] [Google Scholar]
  • 26.Orädd G, Andersson AS, Rilfors L, Lindblom G, Standberg E, Andrén PE. α-Methylene ordering of acyl chains differs in glucolipids and phosphatidylglycerol from Acholeplasma laidlawii membranes: 2H-NMR quadrupole splittlings from individual lipids in mixed bilayers. Biochim Biophys Acta. 2000;468:329–344. doi: 10.1016/s0005-2736(00)00273-x. [DOI] [PubMed] [Google Scholar]
  • 27.Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res. 2011;45:69–86. doi: 10.1159/000324598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Burt BA. Relative consumption of sucrose and other sugars: has it been a factor in reduced caries experience? Caries Res. 1993;27(Suppl):56–63. doi: 10.1159/000261604. [DOI] [PubMed] [Google Scholar]
  • 29.Woodward M, Walker AR. Sugar consumption and dental cries: evidence from 90 countries. Br Dent J. 1994;176:297–302. doi: 10.1038/sj.bdj.4808437. [DOI] [PubMed] [Google Scholar]
  • 30.Tanzer JM, Livingston J, Thompson AM. The microbiology of primary dental caries in humans. J Dent Educ. 2001;65:1028–1037. [PubMed] [Google Scholar]
  • 31.Zeng L, Burne RA. Comprehensive mutational analysis of sucrose-metabolizing pathways in Streptococcus mutans reveals novel roles for sucrose phosphotransferase system permease. J Bacteriol. 2013;195:833–843. doi: 10.1128/JB.02042-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Chiu T-H. Biosynthesis of galactosyl lipids and polysaccharide in Streptococcus mutans. Biochim Biophys Acta. 1988;963:359–366. doi: 10.1016/0005-2760(88)90302-5. [DOI] [PubMed] [Google Scholar]

RESOURCES