Streptococcus suis serotype 2 is an important porcine and human pathogen. Lipoteichoic acid (LTA) from S. suis has been suggested to contribute to its virulence, and absence of d-alanylation from the S. suis LTA is associated with increased susceptibility to cationic antimicrobial peptides. Here, using high-resolution NMR spectroscopy and MS analyses, we characterized the LTA structures from three S. suis serotype 2 strains differing in virulence, sequence type (ST), and geographical origin. Our analyses revealed that these strains possess–in addition to the typical type I LTA present in other streptococci–a second, mixed-type series of LTA molecules of high complexity. We observed a ST-specific difference in the incorporation of glycosyl residues into these mixed-type LTAs. We found that strains P1/7 (ST1, high virulence) and SC84 (ST7, very high virulence) can attach a 1,2-linked α-D-Glcp residue as branching substituent to an α-D-Glcp that is 1,3-linked to glycerol phosphate moieties and that is not present in strain 89-1591 (ST25, intermediate virulence). In contrast, the latter strain could glycosylate its LTA at the glycerol O-2 position, which was not observed in the other two strains. Using LTA preparations from WT strains and from mutants with an inactivated prolipoprotein diacylglyceryl transferase, resulting in deficient lipoprotein acylation, we show that S. suis LTAs alone do not induce Toll-like receptor 2–dependent pro-inflammatory mediator production from dendritic cells. In summary, our study reveals an unexpected complexity of LTAs present in three S. suis serotype 2 strains differing in genetic background and virulence.

NMR, bacteria, cell wall, mass spectrometry, lipoteichoic acid, lipoprotein, Streptococcus suis, bacterial virulence, glycolipid structure

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,2,6,0,3,0,3,0,3,6,0,3,2	aDGlcp	96.5	71.9	73.3	70.0	72.4	61.1
3,2,6,0,3,0,3,0,3,6,0,3	aDGlcp	96.6	75.6	71.8	69.6	71.2	64.6
3,2,6,0,3,0,3,0,3,6,0	x?Gro
3,2,6,0,3,0,3,0,3,6	P
3,2,6,0,3,0	x?Gro	67.0	69.9	62.7
3,2,6,0,3	P
3,2,6,0,3,0,3,0,3,2	aDGlcp
3,2,6,0,3,0,3,0,3	aDGlcp
3,2,6,0,3,0,3,0	x?Gro
3,2,6,0,3,0,3	P
3,2,6,0	x?Gro
3,2,6	P
3,2	aDGlcp
3	aDGlcp	96.6	75.7	71.8	70.2	72.1	61.2
	x?Gro	62.7	71.3	67.0


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,2,6,0,3,0,3,0,3,6,0,3,2	aDGlcp	5.11	3.55-3.60	3.76-3.82	3.43-3.48	3.85-3.93	3.75-3.86
3,2,6,0,3,0,3,0,3,6,0,3	aDGlcp	5.18-5.21	3.68-3.73	3.85-3.90	3.58-3.63	3.80-3.84	4.08-4.17
3,2,6,0,3,0,3,0,3,6,0	x?Gro
3,2,6,0,3,0,3,0,3,6	P
3,2,6,0,3,0	x?Gro	3.88-4.00	4.08-4.12	3.57-3.90
3,2,6,0,3	P
3,2,6,0,3,0,3,0,3,2	aDGlcp
3,2,6,0,3,0,3,0,3	aDGlcp
3,2,6,0,3,0,3,0	x?Gro
3,2,6,0,3,0,3	P
3,2,6,0	x?Gro
3,2,6	P
3,2	aDGlcp
3	aDGlcp	5.17-5.19	3.66-3.70	3.79-3.84	3.44-3.49	3.68-3.73	3.73-3.88
	x?Gro	3.85-3.95	3.89-3.92	3.59-3.70


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,2,6,0,3,0,3,0,3,6,0,3,2	aDGlcp	96.5/5.11	71.9/3.55-3.60	73.3/3.76-3.82	70.0/3.43-3.48	72.4/3.85-3.93	61.1/3.75-3.86
3,2,6,0,3,0,3,0,3,6,0,3	aDGlcp	96.6/5.18-5.21	75.6/3.68-3.73	71.8/3.85-3.90	69.6/3.58-3.63	71.2/3.80-3.84	64.6/4.08-4.17
3,2,6,0,3,0,3,0,3,6,0	x?Gro
3,2,6,0,3,0,3,0,3,6	P
3,2,6,0,3,0	x?Gro	67.0/3.88-4.00	69.9/4.08-4.12	62.7/3.57-3.90
3,2,6,0,3	P
3,2,6,0,3,0,3,0,3,2	aDGlcp
3,2,6,0,3,0,3,0,3	aDGlcp
3,2,6,0,3,0,3,0	x?Gro
3,2,6,0,3,0,3	P
3,2,6,0	x?Gro
3,2,6	P
3,2	aDGlcp
3	aDGlcp	96.6/5.17-5.19	75.7/3.66-3.70	71.8/3.79-3.84	70.2/3.44-3.49	72.1/3.68-3.73	61.2/3.73-3.88
	x?Gro	62.7/3.85-3.95	71.3/3.89-3.92	67.0/3.59-3.70

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

Warning = sub-repeat ending at xXP?: variable unit count (2-9) was assumed to be 2
Warning = sub-repeat ending at xXP?: variable unit count (0-9) was assumed to be 0
O=P(O)(OCC(O)CO[C@H]1O[C@H](COP(=O)(O)OCC(O)CO[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@@H](O)[C@H](O)[C@H]1O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O)OCC(O)COP(=O)(O)OCC(O)COP(=O)(O)OCC(O)COP(=O)(O)OC[C@H]1O[C@H](O[C@H]2[C@@H](OCC(O)CO)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O

1835.23 g/mol (C₅₄H₁₀₃O₅₈P₅)