Structural Reevaluation of Streptococcus pneumoniae Lipoteichoic Acid and New Insights into Its Immunostimulatory Potency

Streptococcus pneumoniae is a Gram-positive human pathogen with a complex lipoteichoic acid (pnLTA) structure. Because the current structural model for pnLTA shows substantial inconsistencies, we reinvestigated purified and, more importantly, O-deacylated pnLTA, which is most suitable for NMR spectroscopy and electrospray ionization-MS spectrometry. We analyzed pnLTA of nonencapsulated pneumococcal strains D39 Delta cps and TIGR4 Delta cps, respectively. The data obtained allowed us to (re) define (i) the position and linkage of the repeating unit, (ii) the putative alpha-GalpNAc substitution at the ribitiol 5-phosphate (Rib-ol-5-P), and (iii) the length of (i. e. the number of repeating units in) the pnLTA chain. We here also describe for the first time that the terminal sugar residues in the pnLTA (Forssman disaccharide; alpha-D-GalpNAc-(1 -> 3)-beta-D-GalpNAc-(1 ->)), responsible for the cross-reactivity with anti-Forssman antigen antibodies, can be heterogeneous with respect to its degree of phosphorylcholine substitution in both O-6-positions. To assess the proinflammatory potency of pnLTA, we generated a (lipopeptide-free) Delta lgt mutant of strain D39 Delta cps, isolated its pnLTA, and showed that it is capable of inducing IL-6 release in human mononuclear cells, independent of TLR2 activation. This finding was quite in contrast to LTA of the Staphylococcus aureus SA113 Delta lgt mutant, which did not activate human mononuclear cells in our experiments. Remarkably, this is also contrary to various other reports showing a proinflammatory potency of S. aureus LTA. Taken together, our study refines the structure of pnLTA and indicates that pneumococcal and S. aureus LTAs differ not only in their structure but also in their bioactivity.