Digestion of Streptococcus pneumoniae cell walls with its major peptidoglycan hydrolase releases branched stem peptides carrying proinflammatory activity

The peptidoglycan of Gram-positive bacteria is known to trigger cytokine re lease from peripheral blood mononuclear cells (PBMCs), However, it requires 100-1000 times more Gram-positive peptidoglycan than Gramnegative Lipopoly saccharide to release the same amounts of cytokines from target cells. Thus , either peptidoglycan is poorly active or only part of it is required for PBMC activation. To test this hypothesis, purified Streptococcus pneumoniae walls were digested with their major autolysin N-acetylmuramoyl-L-alanine amidase, and/or muramidase, Solubilized walls were separated by reverse pha se high pressure chromatography, Individual fractions were tested for their PBMC-stimulating activity, and their composition was determined, Soluble c omponents had a M-r between 600 and 1500, These primarily comprised stem pe ptides cross-linked to various extents. Simple stem peptides (M-r <750) wer e 10-fold less active than undigested peptidoglycan. In contrast, tripeptid es (M-r >1000) were greater than or equal to 100-fold more potent than the native material. One dipeptide (inactive) and two tripeptides (active) were confirmed by post-source decay analysis. Complex branched peptides represe nted less than or equal to 2% of the total material, but their activity (w/ w) was almost equal to that of LPS, This is the first observation suggestin g that peptidoglycan stem peptides carry high tumor necrosis factor-stimula ting activity. These types of structures are conserved among Gram-positive bacteria and will provide new material to help elucidate the mechanism of p eptidoglycan-induced inflammation.