Development of a Listeria monocytogenes EGDe Partial Proteome Reference Map and Comparison with the Protein Profiles of Food Isolates

Abstract

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.

pbp2229 -Mediated Nisin Resistance Mechanism in Listeria monocytogenes Confers Cross-Protection to Class IIa Bacteriocins and Affects Virulence Gene Expression

“…The total cellular proteins of L. monocytogenes 412 and 412C were extracted, resolved by 2-D gel electrophoresis, stained with colloidal Coomassie brilliant blue, and analyzed as described previously (33), except that 18-cm isoelectric focusing strips were used for one-dimensional electrophoresis. Proteins were extracted from two independent cultures of each strain, and at least two 2-D gel electrophoresis analyses were conducted for each sample.…”

Comparative Proteomic Analysis of Listeria monocytogenes Strains F2365 and EGD

“…Previous investigations of the L. monocytogenes proteome have largely used 2D gel electrophoresis followed by identification of individual spots by mass spectrometry (13,16,22,24,45,54). In the current study, we used MuDPIT (2D LC directly inline with ESI and MS-MS) to evaluate the expressed proteome of EGD and F2365.…”

“…MuDPIT analysis was performed on three replicates of EGD and F2365 by using strong cation exchange followed by reverse-phase chromatography coupled directly in line with an electrospray ionization (ESI) ion trap tandem mass spectrometer (LCQ; ThermoElectron Corp.) exactly as previously described (37). The salt gradient applied in this study was applied in steps of 0, 10,15,20,25,30,35,40,45,50,57,64,90, and 700 mM ammonium acetate in 5% acetonitrile-0.1% formic acid. The reverse-phase gradient used 0.1% formic acid in acetonitrile.…”

Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

“…These include the readiness to resume growth at 37°C following starvation at 4°C (1) and the apparent molecular weight of the virulence determinant ActA (21). In addition, the protein profiles of three food-derived isolates of serotype 1/2a were found to have significant differences from those of the serotype 1/2a strain EGDe, the genome of which has been sequenced, suggesting differences either in gene content or in expression (34). Overall, however, limited information currently exists concerning the genomic diversity and virulence of L. monocytogenes strains isolated from routine surveys of food with no known implication in illness.…”