Distinct protein patterns associated with Listeria monocytogenes InlA- or InlB-phagosomes.
Internalization of Listeria monocytogenes into non-phagocytic cells is mediated by the interactions between the two bacterial invasion proteins InlA (internalin) and InlB and their cellular surface receptors E-cadherin and c-Met. To get an insight into all the cellular components necessary for uptake and early intracellular life, we undertook a global proteomic characterization of the early listerial phagosome in the human epithelial cell line LoVo. First, we proceeded to an immunocytochemical characterization of intracellular marker recruitment to phagosomes containing latex beads coated with InlA or InlB. E-cadherin and c-Met were, as expected, rapidly recruited to the phagosomal formation site. Phagosomes subsequently acquired the early endosomal antigen 1 (EEA1) and the lysosomal-associated membrane protein 1 (LAMP1), while presenting a more delayed enrichment of the lysosomal hydrolase cathepsin D. Early phagosomes devoid of lysosomal, endoplasmic reticulum and Golgi enzymatic activities could then be isolated by subcellular fractionation of LoVo cells. Two-dimensional gel electrophoresis (2DPAGE) revealed differences between the protein profiles of InlA- or InlB-phagosomes and those of early/late endosomes or lysosomes of naive LoVo cells. One major protein specifically recruited on the InlB-phagosomes was identified by mass spectrometry as MSF, a previously reported member of the septin family of GTPases. MSF forms filaments that co-localize with the actin cytoskeleton in resting cells and it is recruited to the entry site of InlB-coated beads. These results suggest that MSF is a putative effector of the InlB-mediated internalization of L. monocytogenes into host cells.
Pizarro-Cerdá
Javier
J
Unité des Interactions Bactéries Cellules, Institut Pasteur, 75724 Paris, France.
Jonquières
Renaud
R
Gouin
Edith
E
Vandekerckhove
Joel
J
Garin
Jérome
J
Cossart
Pascale
P
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000032:analysis / Q000378:metabolism; D001426:Bacterial Proteins / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008089:Listeria monocytogenes / Q000737:chemistry / Q000472:pathogenicity; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000494:pharmacology*; D018832:Molecular Chaperones / Q000032:analysis / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000199:Actins; D001426:Bacterial Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; C095768:inlB protein, Listeria monocytogenes; C069761:internalin protein, Bacteria; D000251:Adenosine Triphosphatases