IAI Accepts, published online ahead of print on 13 October 2008

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Infect. Immun. doi:10.1128/IAI.00614-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from vacuole

Jennifer Smith, Joanna Manoranjan, Miao Pan, Amro Bohsali, Junjie Xu, Jun Liu, Kent L. McDonald, Agnieszka Szyk, Nicole LaRonde-LeBlanc, and Lian-Yong Gao^*

Department of Cell Biology and Molecular Genetics and Maryland Pathogens Research Institute, University of Maryland, College Park, MD 20742; Molecular and Cell Biology Graduate Program, University of Maryland, College Park, MD 20742; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Electron Microscope Laboratory, University of California Berkeley, Berkeley, CA 94720; Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742

^* To whom correspondence should be addressed. Email: lygao{at}umd.edu.

The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and WT using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores of 4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.

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