title: Association of ActA to peptidoglycan revealed by cell wall proteomics of intracellular Listeria monocytogenes.
abstract: 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.
authors.affiliation: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: García-del Portillo
authors.forename: Enrique
authors.initials: E
authors.lastname: Calvo
authors.forename: Valentina
authors.initials: V
authors.lastname: D'Orazio
authors.forename: M Graciela
authors.initials: MG
authors.lastname: Pucciarelli
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism* / Q000382:microbiology; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D008089:Listeria monocytogenes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D010455:Peptides / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D010455:Peptides; D011134:Polysaccharides; C072980:actA protein, Listeria monocytogenes