Bacterial membrane lipids are critical for membrane bilayer formation, cell division, protein localization, stress responses, and pathogenesis. Despite their critical roles, membrane lipids have not been fully elucidated for many pathogens. Here, we report the discovery of a novel cationic glycolipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), which is synthesized in high abundance by the bacterium Streptococcus agalactiae (Group B Streptococcus, GBS). To our knowledge, Lys-Glc-DAG is more positively charged than any other known lipids. Lys-Glc-DAG carries 2 positive net charges per molecule, distinct from the widely described lysylated phospholipid lysyl-phosphatidylglycerol (Lys-PG) that carries one positive net charge due to the presence of a negatively charged phosphate moiety. We use normal phase liquid chromatography (NPLC) coupled with electrospray ionization (ESI) high-resolution tandem mass spectrometry (HRMS/MS) and genetic approaches to determine that Lys-Glc-DAG is synthesized by the enzyme MprF in GBS, which covalently modifies the neutral glycolipid Glc-DAG with the cationic amino acid lysine. GBS is a leading cause of neonatal meningitis, which requires traversal of the endothelial blood-brain barrier (BBB). We demonstrate that GBS strains lacking mprF exhibit a significant decrease in the ability to invade BBB endothelial cells. Further, mice challenged with a GBS?mprF mutant developed bacteremia comparably to wild-type (WT) infected mice yet had less recovered bacteria from brain tissue and a lower incidence of meningitis. Thus, our data suggest that Lys-Glc-DAG may contribute to bacterial uptake into host cells and disease progression. Importantly, our discovery provides a platform for further study of cationic lipids at the host-pathogen interface.

group B Streptococcus, mass spectrometry, Streptococcus agalactiae, lipids, cationic glycolipid

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCCCCCC=CCCCCCCCCCC(=O)OC(COC(=O)CCCCCCCCCCCCCCC)COC1O[C@H](COC(=O)C(N)CCCCN)[C@@H](O)[C@H](O)[C@H]1O

885.278 g/mol (C₄₉H₉₂N₂O₁₁)

Yeast is an integral part of mammalian microbiome, and like commensal bacteria, has the potential of being harnessed to influence immunity in clinical settings. However, functional specificities of yeast-derived immunoregulatory molecules remain elusive. Here we find that while under steady state, β-1,3-glucan-containing polysaccharides potentiate pro-inflammatory properties, a relatively less abundant class of cell surface polysaccharides, dubbed mannan/β-1,6-glucan-containing polysaccharides (MGCP), is capable of exerting potent anti-inflammatory effects to the immune system. MGCP, in contrast to previously identified microbial cell surface polysaccharides, through a Dectin1-Cox2 signaling axis in dendritic cells, facilitates regulatory T (Treg) cell induction from naïve T cells. Furthermore, through a TLR2-dependent mechanism, it restrains Th1 differentiation of effector T cells by suppressing IFN-γ expression. As a result, administration of MGCP display robust suppressive capacity towards experimental inflammatory disease models of colitis and experimental autoimmune encephalomyelitis (EAE) in mice, thereby highlighting its potential therapeutic utility against clinically relevant autoimmune diseases.

polysaccharides, medicine, immunity, β-glucan, yeast

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*OC[C@H]1O[C@H](OC[C@H]2O[C@H](OC[C@H]3O[C@H](OC[C@H]4O[C@H](*)[C@@H](O[C@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]5O[C@H]5O[C@H](CO)[C@@H](O)[C@H](O[C@H]6O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]6O)[C@@H]5O)[C@@H](O)[C@@H]4O)[C@@H](O)[C@@H](O)[C@@H]3O)[C@@H](O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O)[C@@H](O)[C@@H]2O)[C@@H](O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O

1621.41 g/mol (C₆₀H₁₀₀R₂O₅₀, R = next and previous repeats, not counted in mol weight)

Yeast is an integral part of mammalian microbiome, and like commensal bacteria, has the potential of being harnessed to influence immunity in clinical settings. However, functional specificities of yeast-derived immunoregulatory molecules remain elusive. Here we find that while under steady state, β-1,3-glucan-containing polysaccharides potentiate pro-inflammatory properties, a relatively less abundant class of cell surface polysaccharides, dubbed mannan/β-1,6-glucan-containing polysaccharides (MGCP), is capable of exerting potent anti-inflammatory effects to the immune system. MGCP, in contrast to previously identified microbial cell surface polysaccharides, through a Dectin1-Cox2 signaling axis in dendritic cells, facilitates regulatory T (Treg) cell induction from naïve T cells. Furthermore, through a TLR2-dependent mechanism, it restrains Th1 differentiation of effector T cells by suppressing IFN-γ expression. As a result, administration of MGCP display robust suppressive capacity towards experimental inflammatory disease models of colitis and experimental autoimmune encephalomyelitis (EAE) in mice, thereby highlighting its potential therapeutic utility against clinically relevant autoimmune diseases.

polysaccharides, medicine, immunity, β-glucan, yeast

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](*)[C@H](O)[C@@H](O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O

486.423 g/mol (C₁₈H₃₀R₂O₁₅, R = next and previous repeats, not counted in mol weight)

Chroogomphus rutilus (CR) possesses anti-inflammatory, antioxidant, and hypoglycemic properties. However, studies are yet to evaluate the anti-osteoporotic activity of the fungi and its polysaccharides. Therefore, this study is aimed at characterizing and evaluating the anti-osteoporotic effects of a novel polysaccharide from CR. The neutral polysaccharide CRP2 extracted and purified from the fruiting body of CR had a molecular weight of 20.41 kDa. Monosaccharide composition analysis revealed that CRP2 is composed of galactose, glucose, fucose, and mannose. The backbone of CRP2 primarily consisted of →6)-α-D-Galp-(1→residues, with specific site substitutions speculated at partial positions, such as O-CH3 substitution at H-3 position, or a branch site located at C-2, including α-L-Fucp-(1→6)-β-D-Glcp-(1→and α-D-Manp-(1→. CRP2 treatment increased trabecular bone density, restored a network-shaped structure, and upregulated the expression of osteoblast differentiation markers, including runt-related transcription factor 2, osterix, osteocalcin, and osteopontin in the femoral tissue of mice with osteoporosis (OP). Additionally, CRP2 treatment suppressed the expression of tumor necrosis factor-α and interleukin-1β in the femoral tissue of mice with OP. Mechanistically, CRP2 exerted anti-OP effect by inhibiting inflammation and promoting osteogenesis through the transforming growth factor β-1/Smad pathway. Conclusively, these findings augment our understanding of the potential role of CRP2 in OP treatment

Structural characterization, Chroogomphus rutilus polysaccharide, osteogenic differentiation, osteoporosis, TGFβ-1/Smad signaling

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
6,6,6	aDGalp	96.81	76.76	67.31	68.44	67.72	65.41
6,6	aDGalp	96.81	67.21	68.94	68.44	67.72	65.41
6,6,6,2	aDManp	101.34	68.94	76.48	65.69	72.29	59.97
6,6,6,2,6	aLFucp	96.89	67.21	68.74	68.23	66.12	14.68
6,6,6,2	bDGlcp	101.91	72.07	74.51	73.73	73.80	65.69
6	aDGalp	96.81	67.21	68.94	68.44	67.72	65.41
3	Me	55.19
	aDGalp	100.41	67.31	77.56	66.44	67.72	65.41


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
6,6,6	aDGalp	4.98	3.76	4.00	3.81	4.14	3.60-3.82
6,6	aDGalp	4.92	3.77	4.00	3.81	4.14	3.60-3.82
6,6,6,2	aDManp	5.03	4.02	3.92	3.60	3.73	3.71-3.82
6,6,6,2,6	aLFucp	4.94	3.77	3.81	3.94	4.08	1.17
6,6,6,2	bDGlcp	4.45	3.24	3.44	3.57	3.61	3.64-3.83
6	aDGalp	4.92	3.77	4.00	3.81	4.14	3.60-3.82
3	Me	3.38
	aDGalp	5.00	3.75	3.51	3.83	4.14	3.60-3.82


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
6,6,6	aDGalp	96.81/4.98	76.76/3.76	67.31/4.00	68.44/3.81	67.72/4.14	65.41/3.60-3.82
6,6	aDGalp	96.81/4.92	67.21/3.77	68.94/4.00	68.44/3.81	67.72/4.14	65.41/3.60-3.82
6,6,6,2	aDManp	101.34/5.03	68.94/4.02	76.48/3.92	65.69/3.60	72.29/3.73	59.97/3.71-3.82
6,6,6,2,6	aLFucp	96.89/4.94	67.21/3.77	68.74/3.81	68.23/3.94	66.12/4.08	14.68/1.17
6,6,6,2	bDGlcp	101.91/4.45	72.07/3.24	74.51/3.44	73.73/3.57	73.80/3.61	65.69/3.64-3.83
6	aDGalp	96.81/4.92	67.21/3.77	68.94/4.00	68.44/3.81	67.72/4.14	65.41/3.60-3.82
3	Me	55.19/3.38
	aDGalp	100.41/5.00	67.31/3.75	77.56/3.51	66.44/3.83	67.72/4.14	65.41/3.60-3.82

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*OC[C@H]1O[C@H](OC[C@H]2O[C@H](OC[C@H]3O[C@H](OC[C@H]4O[C@H](OC[C@H]5O[C@H](OC[C@H]6O[C@H](OC[C@H]7O[C@H](OC[C@H]8O[C@H](*)[C@H](O)[C@@H](OC)[C@H]8O)[C@H](O)[C@@H](O)[C@H]7O)[C@H](O)[C@@H](O)[C@H]6O)[C@H](O[C@H]6O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]6O)[C@@H](O)[C@H]5O)[C@H](O)[C@@H](O)[C@H]4O)[C@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H](O)[C@H]3O)[C@H](O)[C@@H](O)[C@H]2O)[C@H](O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]2O)[C@@H](O)[C@H]1O

1797.578 g/mol (C₆₇H₁₁₂R₂O₅₅, R = next and previous repeats, not counted in mol weight)