The role of ActA in peptidoglycan remodelling of Listeria monocytogenes

Abstract

Listeria monocytogenes is a food-borne bacterial pathogen, which occupies different ecolo-gical niches and is responsible for a number of serious infections in humans. L. monocytogenes has many virulence factors that enable its replication in macrophages and escape from the phagolysosome to the cytoplasm. The actin-assembly inducing protein (ActA) is one of the major virulence factors, which mediates actin polymerization, and thereby aids the intracellular motility and cell-to-cell spread, which is critical for L. monocytogenes pathogenesis. Recently, the ActA protein has been shown to regulate peptidoglycan (PG) biosynthesis during the replication of L. monocytogenes in macrophages. However, the exact mechanism for this phenomenon is unknown. The central hypothesis of the present study is that ActA possesses PG cleaving activity which controls PG biosynthesis and remodelling. ActA is a secreted protein which consists of N-terminal, central and C-terminal domains. Three recombinant His-tagged proteins representing different domains of ActA have been successfully expressed and purified in Escherichia coli C41(DE3). Using a range of approaches, I have shown that the N-terminal domain of ActA has PG cleaving activity. To identify putative catalytic residues, 14 site-directed mutants where acidic residues were replaced with alanine have been generated. None of the mutants resulted in the complete elimination of cleaving activity; however, there was a reduction in activity for two mutants, the MUT 3 and MUT6 proteins. Proteomic analysis revealed the presence of ActA in the culture supernatant and the cell envelope fractions of L. monocytogenes, further supporting the idea that the role of this protein is in cell wall remodelling. ActA has 50 % homology with RpfA, one of the resuscitation-promoting factors found in Mycobacteria. However, domain swapping experiments in which the active domain in RpfA was replaced with the N-terminal domain of ActA, showed limited complementation of the triple Rpf mutant phenotype. The identification of peptidoglycan cleaving activity of the N- terminal domain in ActA from L. monocytogenes would offer a novel opportunity to target and combat the virulence of this significant pathogen.