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Infection and Immunity, December 2008, p. 5478-5487, Vol. 76, No. 12
0019-9567/08/$08.00+0     doi:10.1128/IAI.00614-08
Copyright © 2008, American Society for Microbiology. 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 the Vacuole{triangledown}

Jennifer Smith,1,{dagger} Joanna Manoranjan,1,{dagger} Miao Pan,2 Amro Bohsali,1 Junjie Xu,1 Jun Liu,3 Kent L. McDonald,4 Agnieszka Szyk,5 Nicole LaRonde-LeBlanc,5 and Lian-Yong Gao1,2*

Department of Cell Biology and Molecular Genetics and Maryland Pathogens Research Institute, University of Maryland, College Park, Maryland 20742,1 Molecular and Cell Biology Graduate Program, University of Maryland, College Park, Maryland 20742,2 Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada,3 Electron Microscope Laboratory, University of California Berkeley, Berkeley, California 94720,4 Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 207425

Received 20 May 2008/ Returned for modification 22 June 2008/ Accepted 14 September 2008

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 the wild type by 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 the 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 ~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 the 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.

* Corresponding author. Mailing address: University of Maryland, College Park, 3109 Microbiology Building, College Park, MD 20742. Phone: (301) 405-7562. Fax: (301) 314-9489. E-mail: lygao{at}umd.edu

{triangledown} Published ahead of print on 13 October 2008.

Editor: J. L. Flynn

{dagger} These authors contribute equally to this work.

Infection and Immunity, December 2008, p. 5478-5487, Vol. 76, No. 12
0019-9567/08/$08.00+0     doi:10.1128/IAI.00614-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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