IAI Accepts, published online ahead of print on 9 November 2009

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Infect. Immun. doi:10.1128/IAI.00661-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

THE TETRASPANIN CD81 IS REQUIRED FOR LISTERIA MONOCYTOGENES INVASION

To N. Tham, Edith Gouin, Eric Rubinstein, Claude Boucheix, Pascale Cossart, and Javier Pizarro-Cerdá^*

Institut Pasteur, Unité des Interactions Bactéries-Cellules, Département de Biologie Cellulaire et Infection, Paris F-75015, France; INSERM, U604, Paris F-75015, France; INRA, USC2020, Paris F-75015, France; Institut André Lwoff, Université Paris-Sud, Villejuif F-94807; INSERM, U602, Villejuif F-94807, France

^* To whom correspondence should be addressed. Email: javier.pizarro-cerda{at}pasteur.fr.

Listeria monocytogenes is an intracellular bacterial pathogen that invades epithelial cells by subverting two cellular receptors, E-cadherin and Met. We recently identified type II phosphatidylinositol 4-kinases (PI4KIIs) and as required for bacterial entry downstream of Met. In this work we investigated whether tetraspanins CD9, CD63 and CD81, which figure among the few described molecular partners of the PI4KII, function as molecular adaptors recruiting the PI4KII to the bacterial entry site. We observed by fluorescence microscopy that CD9, CD63 and CD81 are expressed and detected at the cellular surface and also within intracellular compartments, particularly in the case of CD63. In resting cells, colocalization between tetraspanins and the PI4KII is only detectable in restricted areas of the perinuclear region. Upon infection with Listeria, endogenous CD9, CD63 and CD81 were recruited at the bacterial entry site but did not colocalize strictly with endogenous PI4KII. Live cell imaging confirmed that tetraspanins and the PI4KII do not follow the same recruitment dynamics to the Listeria entry site. Depletion of CD9, CD63 and CD81 levels by small interfering RNA (siRNA) demonstrated that CD81 is required for bacterial internalization, identifying for the first time a role for a member of the tetraspanin family in the entry of Listeria within target cells. Moreover, depletion of CD81 inhibits recruitment of PI4KII to the bacterial entry site but not that of the Met receptor, suggesting that this tetraspanin could act as a membrane organizer required for the integrity of signaling events occurring at Listeria entry sites.