Molecular characterization of an autolytic amidase of Listeria monocytogenes EGD

Abstract

The gene encoding a 102 kDa autolysin has been cloned from an expression library of Listeria monocytogenes EGD genomic DNA, using a direct screening protocol. The encoded protein has two domains, an N-terminal enzymic domain showing a high level of homology to the amidase domain of the major autolysin (at/) of Staphylococcus aureus, and a C-terminal, putative cell-wallbinding domain containing four imperfect direct repeats. In order to examine the role of the enzyme, the autolysin-encoding gene was insertionally inactivated by site-specif ic integration of a temperature sensitive plasmid. The enzyme accounts for 66% of the total lytic enzyme activity when L. monocytogenes walls are used as substrate and several of the major autolytic bands are missing on renaturing gels when compared to the wild-type. The enzyme does not appear to be directly involved in cell separation but has a role in motility. Characterization of the recombinant enzyme expressed in Escherichia coli has revealed it to be an amidase and to be able to hydrolyse a range of peptidoglycan substrates.

The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor

“…Abrogation of Ami expression was confirmed by analysing 1% SDS extracts, which are representative of surface protein extracts (Tabouret et al, 1992), by Western blotting using anti-Ami Ami-mediated adhesion 1213 serum. The band at < 100 kDa corresponding to the complete form of Ami (McLaughlan and Foster, 1998) was detected in extracts of parental strains, but not in those of mutants ( Fig. 1).…”

“…The gene immediately downstream from ami, pyrG, is transcribed convergently to it (Braun et al, 1997;McLaughlan and Foster, 1998). Thus, insertional events in ami were unlikely to have a polar effect.…”

“…The ami gene was identified by Southern blotting of L. monocytogenes EGD chromosomal DNA using the 3 H end of the inlB gene as a probe (Braun et al, 1997) and by screening an expression library for clones producing lytic enzymes (McLaughlan and Foster, 1998). Ami of EGD is a 917-amino-acid protein with three characteristic domains: (i) a 30-amino-acid putative signal sequence; (ii) a 179-amino-acid N-terminal domain similar to the alanine amidase domain of the Atl autolysin of S. aureus; (iii) a C-terminal domain (between amino acids 262 and 917) made up of four repeats of approximately 160 amino acids; each repeat can be divided into two modules containing the dipeptide GW (`GW modules'), module 1 consisting of 82±83 amino acids and module 2 of 78 amino acids (Braun et al, 1997).…”

Harnessing Listeria monocytogenes to target tumors

“…The bacterial ligands of these entry receptors identified to date are surface proteins, such as the internalins, InlA and InlB, the actin-polymerizing protein ActA, and p60. Also, putative adhesions have been identified such as surface protein Ami, which is similar to InlB, [29][30][31] and lap, which is involved in attachment to Caco-2 cells. 32 In addition to surface proteins, lectins may be involved in LM adhesion to eukaryotic cells Because of its cytosolic localization, Listeria monocytogenes (LM) has long been considered an attractive tool for delivering tumor-associated antigens (TAA) antigens in vivo to combat cancer.…”

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…33 Ami is a 99 kDa protein exhibiting N-acetylmuramoyl-Lalanine amidase activity. 34 Its N-terminal domain is responsible for cleavage of the amide bond between N-acetylmuramic acid and l-alanine residues in the peptidoglycan. Its C-terminal cell wall-anchoring (CWA) domain contains eight glycine-tryptophan (GW) modules, through which Ami associates to the bacterial surface, putatively by interaction with lipoteichoic acids (LTAs).…”