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.

Switching Lifestyles Is an in vivo Adaptive Strategy of Bacterial Pathogens

“…Thus, different active conformations of ActA might exist in vivo and play a role in deciding the fate of L. monocytogenes. An ActA-mediated lifestyle switch may also be driven by its binding to the peptidoglycan layer during the intracellular phase, although the signal(s) that enable such an association with the cell wall remain unknown (García-del Portillo et al, 2011). Interestingly, a decrease in ActA levels was also found to be correlated with the persistence of L. monocytogenes in vacuoles of non-phagocytic host cells (Kortebi et al, 2017).…”

The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5′-UTR Variant

“…Mutants that lack sortase SrtA and SrtB activity show impaired LPXTG protein anchoring to the peptidoglycan [10] as well as differences in the relative levels of certain LPXTG proteins [11] . Recent studies also showed major changes in the cell wall proteome when L. monocytogenes proliferate inside epithelial cells [12] . Upregulation of defined LPXTG proteins has been observed in intracellular bacteria, including Internalin-A and Lmo0514 [12] .…”

“…Recent studies also showed major changes in the cell wall proteome when L. monocytogenes proliferate inside epithelial cells [12] . Upregulation of defined LPXTG proteins has been observed in intracellular bacteria, including Internalin-A and Lmo0514 [12] . The mechanisms that regulate the coordinated production of such a large number of LPXTG proteins nonetheless remain largely unknown.…”

Three decades of listeriology through the prism of technological advances

“…Future studies, for instance using cryo-electron tomography, may elucidate the stressosome structure in vivo and further assess the contribution of membrane anchor proteins and RsbR paralogues in stressosome activation. Earlier MS-based proteomics studies characterised the Listeria proteome of both extraand intracellular bacteria in detail with a particular focus on the cell wall proteome containing many important virulence factors and proteins that interact with the host (Calvo et al, 2005;Garcia-del-Portillo et al, 2011;Quereda et al, 2013). Together, two decades of Listeria omics profiling by the Cossart laboratory and other groups resulted in more than 600 genome, transcriptome and proteome datasets that are integrated and available on listeriomics.pasteur.fr , an invaluable resource to decipher key regulatory mechanisms of Listeria gene expression by a systems biology approach.…”