AsnB Mediates Amidation of -Diaminopimelic Acid Residues in the Peptidoglycan of and Affects Bacterial Surface Properties and Host Cell Invasion.

A mutant of ScottA with a transposon in the 5' untranslated region of the gene was identified to be hypersensitive to the antimicrobial -cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of -diaminopimelic acid (DAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates DAP amidation in . Deficiency in DAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to DAP its LPXTG motif. Together, this work shows that AsnB of , like several of its homologs in related Gram-positive bacteria, mediates the amidation of DAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan DAP residues in cell wall anchoring of InlA and in bacterial virulence.