Biochemical characterization of evasion from peptidoglycan recognition by Staphylococcus aureusd-alanylated wall teichoic acid in insect innate immunity

We recently reported that d-alanylation of Staphylococcus aureus wall teichoic acid (WTA) mitigates an induction of the Toll-mediated humoral response in Drosophila by interfering with peptidoglycan (PG) recognition by PG recognition protein-SA (PGRP-SA). Here, we investigated the mode of this interference by using an in vitro cell free system from larvae of the coleoptran insect Tenebrio molitor. WTA modification on PG had a potent inhibitory effect on PGRP-SA-mediated Toll proteolytic cascade activation, and the d-alanylation of WTA enhanced its inhibitory effect. Purified d-alanylated WTA released from PG lost its inhibitory action on both Toll cascade activation and PGRP-SA binding to insoluble PG. The inhibition of PGRP-SA binding to PG by d-alanylated WTA took place not only on polymeric PG but also on WTA-attached disaccharide units of monomeric PG. These results suggest that d-alanylation-mediated evasion requires the covalent bonding of d-alanylated WTA to PG, but not net-like cross-linking structure of PG.