Quantitative Proteome Analyses Identify PrfA-Responsive Proteins and Phosphoproteins in Listeria monocytogenes

• Published in: Journal of proteome research 2014-12, Vol.13 (12), p.6046-6057
• Main Authors: Misra, Sandeep Kumar, Moussan Désirée Aké, Francine, Wu, Zongfu, Milohanic, Eliane, Cao, Thanh Nguyen, Cossart, Pascale, Deutscher, Josef, Monnet, Véronique, Archambaud, Cristel, Henry, Céline
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - analysis; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Chromatography, Liquid; Culture Media - chemistry; Culture Media - pharmacology; Glucose - pharmacology; Life Sciences; Listeria monocytogenes - genetics; Listeria monocytogenes - metabolism; Listeria monocytogenes - pathogenicity; Mutation; Peptide Termination Factors - analysis; Peptide Termination Factors - genetics; Peptide Termination Factors - metabolism; Peptides - analysis; Peptides - genetics; Peptides - metabolism; Phosphoproteins - analysis; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation - drug effects; Proteome - analysis; Proteome - genetics; Proteome - metabolism; Proteomics - methods; Purines - biosynthesis; Serine - genetics; Serine - metabolism; Tandem Mass Spectrometry; Threonine - genetics; Threonine - metabolism; Tyrosine - genetics; Tyrosine - metabolism; Virulence - genetics
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr500929u

Protein phosphorylation is a major mechanism of signal transduction in bacteria. Here, we analyzed the proteome and phosphoproteome of a wild-type strain of the food-borne pathogen Listeria monocytogenes that was grown in either chemically defined medium or rich medium containing glucose. We then compared these results with those obtained from an isogenic prfA* mutant that produced a constitutively active form of PrfA, the main transcriptional activator of virulence genes. In the prfA* mutant grown in rich medium, we identified 256 peptides that were phosphorylated on serine (S), threonine (T), or tyrosine (Y) residues, with a S/T/Y ratio of 155:75:12. Strikingly, we detected five novel phosphosites on the virulence protein ActA. This protein was known to be phosphorylated by a cellular kinase in the infected host, but phosphorylation by a listerial kinase had not previously been reported. Unexpectedly, SILAC experiments with the prfA* mutant grown in chemically defined medium revealed that, in addition to previously described PrfA-regulated proteins, several other proteins were significantly overproduced, among them were several proteins involved in purine biosynthesis. This work provides new information for our understanding of the correlation among protein phosphorylation, virulence mechanisms, and carbon metabolism.