Association of ActA to Peptidoglycan Revealed by Cell Wall Proteomics of Intracellular Listeria monocytogenes

Francisco García-Del Portillo,Enrique Calvo,Valentina D'Orazio,M Graciela Pucciarelli

doi:10.1074/jbc.m111.230441

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.

Highlights

Identification of Surface Proteins in Cell Wall of Intracellular L. monocytogenes by High Resolution Mass Spectrometry (MS)— To identify cell wall-associated proteins in intracellular L. monocytogenes, peptidoglycan was isolated at 6 h post-infection from wild-type strain EGDe growing inside human epithelial cells
A surface protein not previously reported to be associated with the peptidoglycan, the actin assembly-inducing protein anchored to the membrane (ActA), and the secreted protein listeriolysin-O (LLO) were identified by proteomics in the peptidoglycan isolated from intracellular bacteria with 19 and 11 unique peptides, respectively (Table 1)
These data suggested that these two proteins, known in extracellular bacteria to be anchored to the membrane (ActA) or secreted (LLO), could associate with the cell wall during the intracellular phase of the infection

Summary

Introduction

Gram-positive bacteria of the genus Listeria contain a large variety of surface proteins that associate with the cell wall (5). Bacteria belonging to this genus carry the largest family of proteins predicted to be covalently anchored to the peptidoglycan upon cleavage of a C-terminal LPXTG sorting motif (3). More than 40 genes encoding LPXTG proteins have been annotated in every Listeria genome sequenced to date (11–13) This feature is shared by nonpathogenic and pathogenic species. Cell Wall Proteome of Intracellular Listeria pathogenic species of the genus, as the intracellular pathogen Listeria monocytogenes (14) Many of these proteins are important virulence factors involved in promoting entry of the bacteria into the host cell Many of these proteins are important virulence factors involved in promoting entry of the bacteria into the host cell (reviewed in Refs. 5, 15, 16)

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