Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that colonizes the cytosol of eukaryotic cells. Recent transcriptomic studies have revealed that intracellular L. monocytogenes alter expression of genes encoding envelope components. However, no comparative global analysis of this cell wall remodeling process is yet known at the protein level. Here, we used high resolution mass spectrometry to define the cell wall proteome of L. monocytogenes growing inside epithelial cells. When compared with extracellular bacteria growing in a nutrient-rich medium, a major difference found in the proteome was the presence of the actin assembly-inducing protein ActA in peptidoglycan purified from intracellular bacteria. ActA was also identified in the peptidoglycan of extracellular bacteria growing in a chemically defined minimal medium. In this condition, ActA maintains its membrane anchoring domain and promotes efficient bacterial entry into nonphagocytic host cells. Unexpectedly, Internalin-A, which mediates entry of extracellular L. monocytogenes into eukaryotic cells, was identified at late infection times (6 h) as an abundant protein in the cell wall of intracellular bacteria. Other surface proteins covalently bound to the peptidoglycan, as Lmo0514 and Lmo2085, were detected exclusively in intracellular and extracellular bacteria, respectively. Altogether, these data provide the first insights into the changes occurring at the protein level in the L. monocytogenes cell wall as the pathogen transits from the extracellular environment to an intracytosolic lifestyle inside eukaryotic cells. Some of these changes include alterations in the relative amount and the mode of association of certain surface proteins.

In follow-up work to this paper, it was found that the identification of an acetylglycerol in the polysaccharide repeating unit is incorrect. The proper placement of the O-acetate group is on ␤-gal C6. The incorrect assignment occurred due to incorrect interpretation of NMR data, which was discovered while working on other polysaccharides that are structurally related to serotype 11A (Calix, J. J., Nahm, M. H., and Zartler, E. R. (2011) J. Bacteriol. 193,[5271][5272][5273][5274][5275][5276][5277][5278]. This correction does not impact the assignment of the other acetyl groups. Also, the correction does not change any genetic and serologic findings described in the paper. The first author, Edward R. Zartler, takes full responsibility for this matter and apologizes for any inconvenience caused. A detailed description of this correction is given in Calix, J. J., Nahm, M. H., and Zartler, E. R. (2011) ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.