The cell wall subproteome of Listeria monocytogenes

Abstract

The surface subproteome of Listeria monocytogenes that includes many proteins already known to be involved in virulence and interaction with host cells has been characterized. A new method for the isolation of a defined surface proteome of low complexity has been established based on serial extraction of proteins by different salts at high concentration, and in all 55 proteins were identified by N-terminal sequencing and mass spectrometry. About 16% of these proteins are of unknown function and three proteins have no orthologue in the nonpathogenic L. innocua and might be involved in virulence mechanisms. Remarkably, a relatively high number of proteins with a function in the cytoplasmic compartment was identified in this surface proteome. These proteins had neither predicted or detectable signal peptides nor could any modification be observed except removal of the N-terminal methionine. Enolase (Lmo2455) is one of these proteins. It was shown to be present in the cell wall of the pathogen by immunoelectron microscopy and, along with heat shock factor DnaK (Lmo1473), elongation factor TU (Lmo2653), and glyceraldehyde-3-phosphate dehydrogenase (Lmo2459), it was found to be able to bind human plasminogen in overlay blots and surface plasmon resonance (SPR) experiments. The KD values of these interactions were determined by SPR measurements. The data indicate a possible role of these proteins as receptors for human plasminogen on the bacterial cell surface. The potential role of this recruitment of a host protease for extracellular invasion mechanisms is discussed.

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

“…sortase substrates (covalently bound to the peptidoglycan) and enzymes related to peptidoglycan metabolism. However, most of the rest of identified proteins were predicted cytosolic proteins that have been largely documented as "moonlighting" proteins also displaying subcellular localization at the cell wall (42). Examples are chaperons (DnaK), elongation factors (EF-Tu), and diverse metabolic enzymes as enolase and glyceraldehyde-3-phosphate dehydrogenase, among others.…”

Improved solubilization of surface proteins from Listeria monocytogenes for 2‐DE

“…The extensive invasive capabilities of L. monocytogenes are principally determined by proteins attached to the surface (cell wall and membrane) of the bacterium by LPXTG motifs, GW modules, hydrophobic tails, lipo-boxes or P60-like domains [3][4][5][6][7][8][9][10][11][12][13][14][15][16]. This notable role of surface proteins in L. monocytogenes virulence and their potential as pharmacological targets [17] make it essential to investigate the proteins in-depth; however, only a small percentage of the characterized surface proteome of L. monocytogenes has been mapped by 2-DE and does not include any proteins attached to the cell surface by LPXTG motifs, GW modules, or hydrophobic tails [18]. This is consistent with proteomic analyses of other Gram-positive bacteria, where examination of 2-D gel maps reveals that cell wall and membrane associated proteins are notably under-represented [17,19,20].…”

Streptococcus pneumoniae ClpL Modulates Adherence to A549 Human Lung Cells through Rap1/Rac1 Activation

“…In contrast, HSP levels in pneumococci are sustained even 1 h after return to normal temperatures (40), suggesting that pneumococcal HSPs may have different roles than those of E. coli. Bacterial HSP60 (GroEL) and HSP 70 (DnaK) are localized at the cell wall or surface and facilitate cell wall synthesis or help the refolding of cell wall proteins (41)(42)(43)(44)(45). Moreover, treatment of Staphylococcus aureus with cefoxitin, a cephalosporin antibiotic, induces GroES, DnaK, and Clp ATPase subunits, such as ClpL (46).…”