Isolation and 2-D-DIGE proteomic analysis of intracellular and extracellular forms of Listeria monocytogenes.
The pathogenicity of Listeria monocytogenes is related to its ability of invading and multiplying in eukaryotic cells. Its main virulence factors are now well characterized, but limited proteomic data is available concerning its adaptation to the intracellular environment. In this study, L. monocytogenes EGD (serotype 1/2a) grown in human THP-1 monocytes (24 h) were successfully separated from host organelles and cytosolic proteins by differential and isopycnic centrifugation. For control, we used cell homogenates spiked with bacteria grown in broth. Proteomes from both forms of bacteria were compared using a 2-D-DIGE approach followed by MALDI-TOF analysis to identify proteins. From 1684 distinct spots, 448 were identified corresponding to 245 distinct proteins with no apparent contamination of host proteins. Amongst them, 61 show underexpression (stress defense; transport systems, carbon metabolism, pyrimidines synthesis, D-Ala-D-Ala ligase) and 22 an overexpression (enzymes involved in the synthesis of cell envelope lipids, glyceraldehyde-3-phosphate, pyruvate and fatty acids). Our proteomic analysis of intracellular L. monocytogenes (i) suggests that bacteria thrive in a more favorable environment than extracellularly, (ii) supports the concept of metabolic adaptation of bacteria to intracellular environment and (iii) may be at the basis of improved anti-Listeria therapy.
Van de Velde
Sébastien
S
Unité de pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium.
Delaive
Edouard
E
Dieu
Marc
M
Carryn
Stéphane
S
Van Bambeke
Françoise
F
Devreese
Bart
B
Raes
Martine
M
Tulkens
Paul M
PM
D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008089:Listeria monocytogenes / Q000302:isolation & purification* / Q000378:metabolism*; D008088:Listeriosis / Q000382:microbiology*; D009000:Monocytes / Q000382:microbiology*; D009711:Nucleotides / Q000378:metabolism; D010453:Peptide Synthases / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013831:Thiamine / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000032:analysis / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D009711:Nucleotides; D020543:Proteome; D014759:Viral Envelope Proteins; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase; D013831:Thiamine