Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole.
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 approximately 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.
Smith
Jennifer
J
Department of Cell Biology and Molecular Genetics and Maryland Pathogens Research Institute, University of Maryland, College Park, Maryland 20742, USA.
Manoranjan
Joanna
J
Pan
Miao
M
Bohsali
Amro
A
Xu
Junjie
J
Liu
Jun
J
McDonald
Kent L
KL
Szyk
Agnieszka
A
LaRonde-LeBlanc
Nicole
N
Gao
Lian-Yong
LY
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism / Q000382:microbiology*; D004912:Erythrocytes / Q000382:microbiology; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D046529:Microscopy, Electron, Transmission; D009165:Mycobacterium Infections, Nontuberculous / Q000378:metabolism / Q000382:microbiology*; D019910:Mycobacterium marinum / Q000378:metabolism / Q000472:pathogenicity*; D012756:Sheep; D014617:Vacuoles / Q000378:metabolism / Q000382:microbiology*
D001426:Bacterial Proteins