Abstract
A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria 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 meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB 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 asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP 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 mDAP residues in cell wall anchoring of InlA and in bacterial virulence.
Highlights
Bacterial cells are surrounded by a rigid peptidoglycan (PG) cell wall whose primary universal function is to maintain cell shape and preserve cell integrity, in hypo-osmotic environments that would otherwise be conducive to cell lysis (Vollmer et al, 2008)
The transcription start site (TSS) of asnB has been reported to be located at −47 bp relative to the start codon in L. monocytogenes EGD-e and at −50 bp in Listeria innocua (Wurtzel et al, 2012), making it likely that transcription of asnB is disrupted in this mutant
T-CIN tolerance was restored to WT level (20-h lag phase) by genetic complementation (Figure 1C), but not by supplementation of the growth medium with Asn (Figure 1D), suggesting that t-CIN sensitivity of the asnB mutant is not related to an Asn deficiency that could be the result of impaired Asn synthetase activity
Summary
Bacterial cells are surrounded by a rigid peptidoglycan (PG) cell wall whose primary universal function is to maintain cell shape and preserve cell integrity, in hypo-osmotic environments that would otherwise be conducive to cell lysis (Vollmer et al, 2008). The tensile strength required for this function is derived from the mesh-like structure of PG, which consists of long polymeric chains of N-acetylglucosamine (GlcNAc), N-acetylmuramic acid (MurNAc) heterodisaccharides, and cross-linked via peptide side chains While this basic architecture has been well conserved, there is a wide variety in PG chemical structures in different bacteria. Differences in PG structure have been observed depending on the growth stage and growth conditions in several bacteria and are developmentally regulated in spore-forming bacteria, where a distinct and unique δ-lactam PG modification exists in the so-called spore cortex, a thick protective PG layer surrounding the germ cell wall which lacks this modification. This allows specific hydrolases embedded on the spore’s surface to selectively cleave the cortex without compromising the germ cell wall upon spore germination
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